Synthesis and evaluation of furoxan-based nitric oxide-releasing derivatives of tetrahydroisoquinoline as anticancer and multidrug resistance reversal agents

Article abstract of DOI:10.1016/j.bmcl.2011.07.077

Multidrug resistance in tumor cells poses a major obstacle to efficient chemotherapy. Several types of agents have been recognized as multidrug resistance inhibitors, among which the tetrahydroisoquinolines is the most studied. In current study 16 furoxan-based nitric oxide-releasing derivatives of tetrahydroisoquinoline were synthesized. Their cytotoxic activities and effects in reversing multidrug resistance have been evaluated. The results revealed that these compounds had moderate cytotoxic effects. Compounds 7a-f, 7h, and 7l showed higher cytotoxicities than the rest, but lower than adriamycin on K562 cell line. Compounds 7d, 7f, and 7l exhibited potent MDR reversal activities on K562/A02 cell line. The accumulation assay indicated that compounds 7d, 7f, and 7l significantly increased the intracellular accumulation of rhodamine123 in K562/A02 cells. Furthermore, these three compounds produced high concentrations of NO in K562/A02 cells. Potentially, the high concentrations of NO produced by NO donor moieties will lead to an increased cytotoxicity to K562/A02 cells. Our results suggested that compounds 7d, 7f, and 7l had anticancer effects, as well as multidrug resistance reversal effects.

Full text of DOI:10.1016/j.bmcl.2011.07.077

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5934–5938
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of furoxan-based nitric oxide-releasing derivatives
of tetrahydroisoquinoline as anticancer and multidrug resistance
reversal agents
Zhi-hong Zou a,b, , Xiao-bu Lan a,c, , Hai Qian a, , Wen-long Huang a, , Yun-man Li d
⇑
⇑
a Centre of Drug Discovery, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China
b Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Southeast University, Nanjing 210009, China
c Department of Organic Chemistry and Medicinal Chemistry, Guangxi Medical University, 22 Shuangyong Road, Nanning, Guangxi 530021, China
d Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Multidrug resistance in tumor cells poses a major obstacle to efficient chemotherapy. Several types of
agents have been recognized as multidrug resistance inhibitors, among which the tetrahydroisoquino-
lines is the most studied. In current study 16 furoxan-based nitric oxide-releasing derivatives of tetrahy-
droisoquinoline were synthesized. Their cytotoxic activities and effects in reversing multidrug resistance
have been evaluated. The results revealed that these compounds had moderate cytotoxic effects. Com-
pounds 7a–f, 7h, and 7l showed higher cytotoxicities than the rest, but lower than adriamycin on
K562 cell line. Compounds 7d, 7f, and 7l exhibited potent MDR reversal activities on K562/A02 cell line.
The accumulation assay indicated that compounds 7d, 7f, and 7l significantly increased the intracellular
accumulation of rhodamine123 in K562/A02 cells. Furthermore, these three compounds produced high
concentrations of NO in K562/A02 cells. Potentially, the high concentrations of NO produced by NO donor
moieties will lead to an increased cytotoxicity to K562/A02 cells. Our results suggested that compounds
7d, 7f, and 7l had anticancer effects, as well as multidrug resistance reversal effects.
Received 26 May 2011
Revised 19 July 2011
Accepted 22 July 2011
Available online 29 July 2011
Keywords:
Multidrug resistance
Anticancer
Tetrahydroisoquinoline
Nitric oxide
Ó 2011 Elsevier Ltd. All rights reserved.
Over the last 30 years, multidrug resistance in tumor cells poses a
major obstacle to efficient chemotherapy and has become a serious
medical concern. One major cause of this phenomenon is due to
over-expression of P-glycoprotein (P-gp, the product of the MDR1
gene). P-gp, which belongs to the adenosine triphosphate (ATP)-
binding cassette (ABC) super-family of transporters,1,2 has been
likened to a molecular ‘hydrophobic vacuum cleaner’, extracting
substrates within the cells and repelling them to promote MDR.3
In the last decades, several P-gp inhibitors or modulators, notable
among which are verapamil, HZ08(a derivative of THIQ without
furoxan moiety, found in our laboratory, has been shown to possess
MDR reversal activity, Fig. 1),4 tariquidar and W198 (Fig. 1),5–7 have
been investigated for their potential as MDR reversal agents in anti-
cancer chemotherapies. These agents have been traditionally char-
acterized by their strong potency to inhibit P-gp with low
cytotoxic profiles. Among these compounds, tetrahydroisoquinoline
(THIQ) derivatives are identified as a class of promising MDR rever-
sal agents. However, they need to be applied along with other
chemotherapeutic agents in the treatment of cancer. Meanwhile,
nitric oxide (NO)-based anticancer agents have been developed for
their potential application for cancer chemotherapy at clinic.8
Kotamraju et al. reported that NO was in part responsible for the
pro-apoptotic and anticancer effects of statins that enhanced NO
concentration in MCF-7 cells.9 Furoxans, an important class of NO
donors, can produce high concentrations of NO and exhibit strong
anticancer activities.10 Thus, in this study we designed a series of
furoxan-based NO-releasing derivatives of THIQ as anticancer and
MDR reversal agents. These agents were expected to enhance the
sensitivity of drug-resistant tumor cells to chemotherapeutic agents
and decrease the growth of tumor cells.
A general strategy for the synthesis of the target compounds 7
a–p is shown in Scheme 1. Four phenylsulfonyl-substituted furox-
ans (1a–d) were synthesized according to the literatures.11,12 Sub-
sequently, compounds 1a–d were further reacted with succinic
anhydride in the presence of dicyclohexylcarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP) to form 2a–d. The 1-substituted
derivatives 6a-d were prepared from 3,4-dimethoxyphenethyl-
amine (3) following these literature13–15 procedure. Finally, com-
pounds 6a–d were reacted with 2a–d in the presence of DCC and
DMAP to afford 7a–p.16 The compounds obtained were shown in
Table 1.
⇑
Corresponding authors. Tel.: +86 25 83271302; fax: +86 25 83271480.
E-mail addresses: qianhai24@163.com (H. Qian), ydhuangwenlong@126.com
(W.-l. Huang).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.077

Z.-h. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5934–5938
5935
CN
H3CO
N
H3CO
H3CO
OCH3
OCH3
N
NHC8H17-n
CN
H3CO
N
CH3
OCH3
OCH3
verapamil
HZ08
OCH3
OCH3
Br
O
O
N
O
O
O
N
H3CO
H3CO
N
N
H
NH
O
O
O
N
Tariquidar
W198
Figure 1. Structures of MDR reversal agents.
O
O
N
O
N
O
O
N
N
a
R1
R1
HOOC
O
O
HO
O
SO2Ph
SO2Ph
1a-d
2a-d
f
H3CO
H3CO
O
N
O
O
N
H3CO
H3CO
R1
N
O
O
SO2Ph
NH2
R2
b
O
3
7a-p
c
H3CO
H3CO
e
H3CO
H3CO
N
NH
d
R2
R2
H3CO
H3CO
5a-d
6a-d
NH
R2
O
R2
R1
4b-d
H
1a, 2a
CH2C CCH2
5a, 6a
4b, 5b, 6b
4c, 5c, 6c
4d, 5d, 6d
O
O
1b, 2b
1c, 2c
1d, 2d
CH2CH2
O
CH2(CH2)2CH2
CH2(CH2)4CH2
Scheme 1. Preparation of THIQ derivatives. Reagents and conditions: (a) succinic anhydride, DMAP, DCM, reflux, 8–10 h, 26.4–59.9%; (b) formaldehyde, reflux, 30 min, 70%
for 5a; (c) R2CH2COOH, N2, 190 °C, 3 h, 71–92%; (d) POCl3, toluene, N2, reflux, 2.5 h, 97–98%; (e) KBH4, diethylamine, MeOH, rt, 24 h, 95–99%; (f) DCC, DMAP, DCM, rt, 4 h.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT) colorimetric assay was used to evaluate the effect of
target compounds on the human erythroleukemia K562 cell and
its adriamycin-selected K562/A02 cell, which could keep the adri-
amycin away from the cells.17–22 These cell lines were cultured in
essential medium containing 10% fetal bovine serum at 37 °C in the
presence of 5% CO2. All cells were grown in drug-free culture med-
ium for >14 days before assay. The tested compounds (7a–p, adri-
amycin, HZ08 and verapamil) were dissolved in DMSO (stock
All compounds 7a–p were evaluated for their cytotoxicities and
MDR reversal activities. The results were summarized in Table 2
and Table 3, respectively. The data, as shown in Table 2, revealed
that most of these compounds exerted cytotoxic activities in differ-
ent degrees. Compounds 7a–f, 7h, and 7l displayed higher cyto-
toxic activities than the rest, but lower than adriamycin on K562
cells. Furthermore, in K562 cells, these analogs in combination
with adriamycin (Table 3) exhibited more potent cytotoxic activi-
ties in comparison with those compounds treated alone (Table
2). In conclusion, these compounds might display synergistic cyto-
toxicities with adriamycin in tumor cells. Besides, the cytotoxici-
ties of most compounds, except 7k, displayed differences with
adriamycin on K562/A02 cells, shown in Table 2. The differences
solution, 10 lM) and used within 7 days. The final DMSO concen-
tration never exceeded 0.1% (v/v) either in controls. The concentra-
tions required to inhibit growth by 50% (IC50 values) were
calculated from the cytotoxicity curves by the method of Bliss.23

5936
Z.-h. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5934–5938
Table 1
Table 3
Synthesized compounds
Effect of tested compounds on reversing P-gp-mediated drug resistance
H3CO
O
Compound
IC50 SD, l
M (fold reversal)b
K562/A02
N
O
O
N
R1
K562
N
O
O
H3CO
Adriamycin
+Verapamila
+7a
+7b
+7c
+7d
+7e
+7f
+7g
+7h
+7i
+7j
+7k
+7l
+7m
+7n
+7o
+7p
0.417 0.075(1.00)
0.432 0.135(0.96)
0.253 0.06* (1.65)
0.253 0.031* (1.65)
0.241 0.027* (1.73)
0.224 0.09* (1.86)
0.311 0.14(1.34)
0.284 0.033* (1.47)
0.271 0.02* (1.54)
0.262 0.023* (1.59)
0.278 0.004* (1.50)
0.379 0.13(1.10)
0.393 0.15(1.06)
0.284 0.015* (1.47)
0.383 0.11(1.09)
0.309 0.014(1.35)
0.348 0.02(1.20)
0.309 0.045(1.35)
19.02 0.018(1.00)
SO2Ph
Ph= phenyl
R2
O
1.09 0.014(17.45)
1.881 0.004* (10.11)
3.366 0.12* (5.65)
10.994 0.092* (1.73)
0.594 0.22* (32.00)
1.782 0.021* (10.67)
0.892 0.013* (21.33)
1.782 0.014* (10.67)
9.415 0.033* (2.02)
10.281 0.34* (1.85)
10.117 0.114* (1.88)
11.321 0.021* (1.68)
1.089 0.06* (17.45)
5.943 0.02* (3.20)
9.704 0.23* (1.96)
6.038 0.15* (3.15)
3.766 0.08* (5.05)
Compound
R1
R2
Yield (%)
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
–CH2C„CCH2–
–CH2C„CCH2–
–CH2C„CCH2–
–CH2C„CCH2–
–CH2CH2–
H
38
44
42
39
48
47
52
43
59
50
58
59
29
32
29
34
3,4-Dimethoxybenzyl
Phenoxymethyl
-Naphthylmethyl
a
H
–CH2CH2–
–CH2CH2–
–CH2CH2–
3,4-Dimethoxybenzyl
Phenoxymethyl
a-Naphthylmethyl
–CH2 (CH2)2CH2–
–CH2 (CH2)2CH2–
–CH2 (CH2)2CH2–
–CH2 (CH2)2CH2–
–CH2 (CH2)4CH2–
–CH2 (CH2)4CH2–
–CH2 (CH2)4CH2–
–CH2 (CH2)4CH2–
H
3,4-Dimethoxybenzyl
Phenoxymethyl
-Naphthylmethyl
a
H
a
3,4-Dimethoxybenzyl
Phenoxymethyl
-Naphthylmethyl
Verapamil (as a positive reference drug) and compounds final concentrations
were defined at 10 M.
The fold reversal of MDR = IC50 (adriamycin)/IC50 (tested compound).
Student’s t test, P < 0.05 versus that obtained in the absence of tested compounds.
l
b
a
*
Table 2
Cytotoxicity of tested compounds on K562 and K562/A02 in vitro
compared to others. However, while replacing butynyl group with
other straight-chain alkanes (ethyl, butyl and hexyl), the com-
pounds showed a slight loss of activities. In addition, it should be
Compound
IC50 SD, l
Ma
noted that derivatives with a-naphthylmethyl moiety at C-1 posi-
K562
K562/A02
tion, such as 7d, 7h, 7l, and 7p, showed higher cytotoxicities than
those containing the same substituent at C-2 position. It was im-
plied that the compound bearing a rigid aromatic moiety at C-1
of THIQ increased its cytotoxic activity to tumor cells.
Adriamycin
0.425 0.002
0.901 0.045*
0.905 0.044*
0.892 0.015*
0.875 0.034*
1.148 0.12*
1.087 0.149*
1.451 0.18*
1.032 0.034*
1.558 0.011*
1.933 0.243*
1.502 0.028*
1.004 0.054*
1.734 0.102*
1.65 0.105*
1.794 0.067*
1.604 0.094*
NDb
17.84 0.065
8.079 0.072*
8.055 0.095*
6.362 0.01*
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
HZ08
7.503 0.005*
12.703 0.005*
11.251 0.132*
10.806 0.087*
9.984 0.048*
11.782 0.051*
13.032 0.16*
17.533 0.204
12.192 0.158*
14.599 0.194*
12.511 0.233*
13.475 0.234*
12.292 0.302*
>500
In Table 3, compound 7d with
a-naphthylmethyl group at the
C-1 position of THIQ possessed more efficacy than 7f with 3,
4-dimethoxybenzyl group at the same position. The result implied
that the introduction of
a-naphthylmethyl group at the C-1 posi-
tion of THIQ benefited to improve the MDR reversal activity profile
of the tested compounds. Moreover, the type and length of the
linkers, which connected NO donor moiety to the C-2 position of
THIQ, did not obviously affect the reversal activities. It was worthy
of mention that 7d significantly increased the cytotoxicity of adri-
amycin, with IC50 0.594 lM (32-fold sensitization), in K562/A02
cells. In contrast, 7d only produced a 1.86-fold sensitization to
adriamycin in the parental K562 cells. It indicated that a rigid sub-
stituent at C-1 position of THIQ was important to the increase of
the MDR reversal activity.
a
Cell survival was determined by MTT assay as described previously. IC50
compound concentration required to inhibit tumor cell growth by 50%; the data
were presented as mean SD.
P < 0.05, K562 cells treated with tested compounds compared with reference
compound adriamycin, Student’s t test.
,
*
Most derivatives of THIQ, such as tariquidar, W198 and HZ08,
were identified as inhibitors of P-gp in MDR reversal research.
We next determined whether the reversal activities of target com-
pounds were caused by direct interaction with the transporters.
Therefore, the rhodamine123 accumulation assay was carried out
to determine the effect of compounds 7d, 7f, and 7l on the func-
tional of P-gp. The uptake of rhodamine123 in cells was followed
by monitoring the fluorescence signal with the method de-
scribed.24–26 The data showed that fluorescence value of com-
pounds 7d, 7f, and 7l was increased obviously compared to
control group and shared a similar potency to verapamil group in
K562/A02 cell line ( Fig. 2). The result demonstrated that the
MDR reversal effects probably resulted from the inhibition of
P-gp in K562/A02 cells after the treatment with tested compounds,
particularly 7d.
b
ND, not determined.
might be due to their cytotoxicities or/and MDR reversal activities.
As shown in Table 3, some compounds, especially 7d, 7f, and 7l,
produced a similar or significant sensitization of K562/A02 cells
to adriamycin as compared to the reference agent verapamil.
Moreover, the sensitive effects (presented as fold reversal) induced
by tested compounds were compared in the presence or absence of
adriamycin. The results showed that compounds 7d, 7f, and 7l dra-
matically increased the sensitivity of drug-resistant cells to adria-
cymin, which confirmed that 7d, 7f, and 7l effectively reversed
MDR in adriamycin-selected K562/A02 cell. The analysis above
suggested that compounds 7d, 7f, and 7l had an anticancer effect
as well as a multidrug resistance reversal effect on leukemic cells,
and were therefore considered worthy of further investigation.
In Table 2, compounds 7a–d, with a butynyl group as an alter-
native linker, exhibited more potent cytotoxic activities as
Furthermore, analysis of the cytotoxicities and MDR reversal
activities (as above) indicated that part of these compounds acted
as anticancer and MDR reversal agents. Hence, we assessed
whether the concentration of NO production by the indicated

Z.-h. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5934–5938
5937
4500
4000
3500
3000
2500
2000
1500
1000
500
likely mediated by high concentration of NO produced from furox-
ans. For good anticancer and MDR reversal activities, a rigid aro-
#
*
matic ring, such as
a-naphthylmethyl group, presents at C-1
position on THIQ, whereas a rigid linker between THIQ and furox-
ans significantly increased the cytotoxicities. The rigid aromatic
group at C-1 also improves MDR reversal activities against multi-
drug-resistant K562/A02 cells. Our findings show that compounds
7d, 7f, and 7l, which have dual activities, are promising. Further
biological evaluation, pharmacokinetics study, long-term toxicity,
and mechanistic studies on these three compounds will be re-
ported in due course.
*
*
*
0
control
Verapamil
7d
7f
7l
Group
Acknowledgments
Figure 2. Effects of reversal agents on rhodamine123 accumulation in K562/A02
cells. Tested compounds final concentrations were defined at 10
M. ⁄P < 0.05,
K562/A02 cells treated with tested compounds compared with control group,
Student’s t test. #P < 0.05, K562/A02 cells treated with tested compounds compared
with verapamil group, Student’s t test.
The work was supported by the Important National Science and
Technology Specific Projects (2009ZX09102-033), the National
Natural Science Foundation of China (No. 30772647) and the High
Technology Programs of Jiangsu province (BG2007612).
l
References and notes
1. Juliano, R. L.; Ling, V. Biochim. Biophys. Acta 1976, 455, 152.
2. Sharom, F. J. Pharmacogenomics 2008, 9, 105.
3. Germann, U. A.; Pastan, I.; Gottesman, M. M. Semin. Cell Biol. 1993, 4, 63.
4. Li, Y.; Zhang, H. B.; Huang, W. L.; Li, Y. M. Bioorg. Med. Chem. Lett. 2008, 18,
3652.
5. Krishna, R.; Mayer, L. D. Eur. J. Pharm. Sci. 2000, 11, 265.
6. Klinkhammer, W.; Muller, H.; Globisch, C.; Pajeva, I. K.; Wiese, M. Bioorg. Med.
Chem. 2009, 17, 2524.
7. Thomson-Pharma-Database. http://www.thomson-pharma.com, updated to 10
march, 2011.
8. Siemens, D. R.; Heaton, J. P.; Adams, M. A.; Kawakami, J.; Graham, C. H. Urology
2009, 74, 878.
9. Kotamraju, S.; Williams, C. L.; Kalyanaraman, B. Cancer Res. 2007, 67, 7386.
10. Maksimovic-Ivanic, D.; Mijatovic, S.; Harhaji, L.; Miljkovic, D.; Dabideen, D.;
Fan Cheng, K.; Mangano, K.; Malaponte, G.; Al-Abed, Y.; Libra, M.; Garotta, G.;
Nicoletti, F.; Stosic-Grujicic, S. Mo.l Cancer Ther. 2008, 7, 510.
11. Li, R. W.; Zhang, Y. H. Acta Pharm. Sin. 2001, 36, 821.
12. Moharram, S.; Zhou, A.; Wiebe, L. I.; Knaus, E. E. J. Med. Chem. 2004, 47, 1840.
13. Manske, R. H. Chem Rev. 1942, 1942, 145.
Figure 3. The concentrations of NO produced by 7d, 7f, 7l were correlated with
their cytotoxicities against K562/A02 cells. Cytotoxicities were expressed as IC50
.
The concentration of NO was characterized by its concentration determined in the
cells. Data showed mean SD of individual groups from three separated experi-
ments. The cells treated with HZ08 alone did not affect their growth and did not
release NO (data not shown). The control group treated without agent did not
induce a detectable concentration of NO (data not shown). Statistical analysis was
performed using one-way ANOVA followed by the post hoc Tukey’s test.
14. Buck, J. S. J. Am. Chem. Soc. 1934, 56, 2799.
15. Huang, W.; He, L.; Peng, S. Chin. J. Med. Chem. 1999, 9, 26.
16. Spectroscopic data for target compounds. Compound 7a: 1H NMR (CDCl3,
300 MHz,
d ppm): 2.75 (t, 2H, J = 6.6 Hz, NOCCH2CH2COO), 2.78 (t, 2H,
J = 6.6 Hz, NOCCH2CH2COO), 2.84 (t, 2H, J = 3.5 Hz, C4-H), 3.68 (t, 2H,
J = 3.5 Hz, C3-H), 3.84, (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.58 (s, 2H,
COOCH2C„CCH2O), 4.70 (s, 2H, COOCH2C„CCH2O), 5.09 (s, 2H, C1–H), 6.60
(s, 1H, C5–H), 6.61 (s, 1H, C8–H), 7.60–8.09 (m, 5H, Ph-H); IR (KBr): 2936, 2859,
1745, 1710, 1645, 1620, 1549, 1517, 1454 cmÀ1; MS (ESI, m/z): 586.1([M+H]+,
base peak); Anal.Calcd. for C27H27N3O10S: C, 54.54; H, 4.75; N, 7.07. Found: C,
54.55; H, 4.74; N, 7.03.
compounds in human cancer cell was associated with their
cytotoxicities. The concentrations of nitrite/nitrate produced in
the lysates of K562/A02 cells were characterized by the Griess
assay (Fig. 3).27 As expected, treatment with HZ08 produced little
nitrite/nitrate in the tested cells. And the control group treated
without any agent did not release a detectable concentration of ni-
trite/nitrate in the tested cells. In contrast, treatment with 7d, 7f,
and 7l promoted high concentrations of nitrite/nitrate production.
Figure 3 also revealed that cytotoxic activities relatively increased
as the increasing concentration of NO, suggesting that furoxan
moiety in the tested compound promoted concentration of NO pro-
duction, which was responsible for their strong cytotoxicities
against K562/A02 cells in vitro.
In summary, sixteen THIQ derivatives containing furoxans were
synthesized and their biological activities in vitro were evaluated.
Some compounds exhibited moderate anticancer activities against
K562 and K562/A02 cells. The cytotoxicities of compounds 7a–f,
7h, and 7l were higher than the rest, but lower than adriamycin
on the K562 cell line. Compounds 7d, 7f, and 7l displayed compa-
rable MDR reversal activities with that of reference verapamil on
K562/A02 cell line. These results indicated that 7d, 7f, and 7l prob-
ably had dual activities, an anticancer effect and a multidrug resis-
tance reversal effect. The result of rhodamine123 accumulation
assay demonstrated that compounds 7d, 7f, and 7l probably
blocked pump efflux function of P-gp. Furthermore, NO assay indi-
cated that the potent cytotoxicities of tested compounds were
Compound 7b: 1H NMR (CDCl3, 300 MHz,
d ppm): 2.68 (t, 2H, J = 6.5 Hz,
NOCCH2CH2COO), 2.76 (t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.98–3.14 (m, 4H,
C4–H, ArCH2), 3.41–3.44 (m, 2H, C3–H), 3.84 (s, 6H, 2ÂOCH3), 3.86 (s, 6H,
2ÂOCH3), 4.56 (s, 2H, COOCH2C„CCH2O), 4.69 (s, 2H, COOCH2C„CCH2O), 5.58
(t, 1H, J = 7.5 Hz, C1–H), 6.18–6.21 (m, 3H, Ar-H), 6.60 (s, 1H, C5–H), 6.61 (s, 1H,
C8–H), 7.60–8.08 (m, 5H, Ph-H); IR (KBr): 2931, 2844, 1740, 1622, 1548, 1514,
;
1450 cmÀ1 MS (ESI, m/z): 736.0 ([M+H]+, base peak); Anal.Calcd. for
C
36H37N3O12S: C, 58.77; H, 5.07; N, 5.71. Found: C, 58.68; H, 5.08; N, 5.73.
Compound 7c: 1H NMR (CDCl3, 300 MHz,
d
ppm): 2.70 (t, 2H, J = 6.5 Hz,
NOCCH2CH2COO), 2.75 (t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.78 (t, 2H,
J = 3.3 Hz, C4–H), 2.90–3.22 (m, 2H, C3–H), 3.78–3.83 (d, 2H, J = 7.5 Hz,
PhOCH2), 3.85(s, 3H, OCH3), 3.87 (s, 3H, OCH3), 4.75 (s, 2H,
COOCH2C„CCH2O), 4.77 (s, 2H, COOCH2C„CCH2O), 5.72 (t, 1H, J = 7.5 Hz,
C1–H), 6.64 (s, 1H, C5–H), 6.65 (s, 1H, C8–H), 7.62–8.09 (m, 10H, Ph-H); IR
(KBr): 2933, 2870, 1745, 1645, 1620, 1585, 1548, 1518, 1452 cmÀ1; MS (ESI, m/
z): 692.2 ([M+H]+, base peak); Anal.Calcd. for C34H33N3O11S: C, 59.04; H, 4.81;
N, 6.07. Found: C, 59.09; H, 4.89; N, 6.11.
Compound 7d: 1H NMR (CDCl3, 300 MHz,
d ppm): 2.75 (t, 2H, J = 6.5 Hz,
NOCCH2CH2COO), 2.84 (t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 3.28 (t, 2H,
J = 3.6 Hz, C4–H), 3.67–3.71 (m, 2H, C3–H), 3.38–3.40 (d. 2H, J = 7.5 Hz,
ArCH2), 3.72 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.79 (s, 2H,
COOCH2C„CCH2O), 5.07 (s, 2H, COOCH2C„CCH2O), 5.57 (t, 1H, J = 7.5 Hz,
C1–H), 6.64 (s, 1H, C5–H), 6.66 (s, 1H, C8–H), 7.62–8.09 (m, 12H, Ph-H); IR
(KBr): 2938, 1743, 1635, 1613, 1542, 1518, 1451 cmÀ1; MS (ESI, m/z): 726.2
([M+H]+, base peak); Anal.Calcd. for C38H35N3O10S: C, 62.89; H, 4.86; N, 5.79.
Found: C, 62.84; H, 4.87; N, 5.83.
Compound 7e: 1H NMR (CDCl3, 300 MHz,
d ppm): 2.67 (t, 2H, J = 6.5 Hz,
NOCCH2CH2COO), 2.78 (t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.82 (t, 2H,
J = 3.6 Hz, C4–H), 3.47–3.71 (m, 2H, C3–H), 3.82 (s, 3H, OCH3), 3.84 (s, 3H,

5938
Z.-h. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5934–5938
OCH3) 4.55 (t, 2H, J = 6.6 Hz, COOCH2CH2O), 4.62 (t, 2H, J = 6.6 Hz,
COOCH2CH2O), 4.69 (s, 2H, C1–H), 6.59 (s, 1H, C5–H,), 6.61 (s, 1H, C8–H),
ArCH2), 3.38–3.42(m, 1H, C3–H), 3.61–3.74 (m, 1H, C3–H), 3.82 (s, 6H, 2ÂOCH3),
3.86 (s, 6H, 2ÂOCH3), 4.04 (t, 2H, J = 6.4 Hz, COOCH2(CH2)4CH2O), 4.12 (t, 2H,
J = 6.5 Hz, COOCH2(CH2)4CH2O), 5.57 (t, 1H, J = 7.5 Hz, C1–H), 6.17–6.20 (m, 3H,
Ar-H), 6.61 (s, 1H, C5–H), 6.62 (s, 1H, C8–H), 7.60–8.07 (m, 5H, Ph-H); IR (KBr):
2939, 2859, 1731, 1637, 1616, 1551, 1514, 1448 cmÀ1; MS (ESI, m/z): 768.1
([M+H]+, base peak); Anal.Calcd. for C38H45N3O12S: C, 59.44; H, 5.91; N, 5.47.
Found: C, 59.41; H, 5.93; N, 5.45.
7.59–8.08 (m, 5H, Ph-H); IR (KBr): 2926, 2850, 1738, 1622, 1553, 1513,
;
1449 cmÀ1 MS (ESI, m/z): 562.2 ([M+H]+, base peak); Anal.Calcd. for
C
25H27N3O10S: C, 53.47; H, 4.85; N, 7.48. Found: C, 53.44; H, 4.43; N, 7.45.
Compound 7f: 1H NMR (CDCl3, 300 MHz,
d
ppm): 2.74(t, 2H, J = 6.7 Hz,
NOCCH2CH2COO), 2.82 (t, 2H, J = 6.7 Hz, NOCCH2CH2COO), 2.96 (t, 2H,
J = 3.6 Hz, C4–H), 3.42–3.71 (m, 2H, C3–H), 3.78 (d. 2H, J = 7.5 Hz, ArCH2),
3.85 (d, 12H, 4ÂOCH3), 4.53 (t, 2H, J = 6.6 Hz, COOCH2CH2O), 4.63 (t, 2H,
J = 6.6 Hz, COOCH2CH2O), 5.57 (t, 1H, J = 7.5 Hz, C1–H), 6.17–6.20 (m, 3H, Ar-H),
6.60 (s, 1H, C5–H), 6.61 (s, 1H, C8–H), 7.60–8.08 (m, 5H, Ph-H); IR (KBr): 3462,
1733, 1622, 1553, 1513, 1448 cmÀ1; MS (ESI, m/z): 734.1 ([M+Na]+, base peak);
Anal.Calcd. for C34H37N3O12S: C, 57.38; H, 5.24; N, 5.90. Found: C, 57.40; H,
5.23; N, 5.25.
Compound 7o: 1H NMR (CDCl3, 300 MHz, d ppm): 1.47–1.70 (m, 10H, C4–H,
OCH2(CH2)4CH2O), 2.71 (t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.76 (t, 2H,
J = 6.5 Hz, NOCCH2CH2COO), 2.91 (t, 2H, J = 3.4 Hz, C4–H), 3.00–3.20 (m, 2H,
C3–H), 3.75–3.80 (d, 2H, J = 7.2 Hz, PhOCH2), 3.86 (s, 3H, OCH3), 3.87(s, 3H,
OCH3), 4.09 (t, 2H, J = 6.4 Hz, COOCH2(CH2)4CH2O), 4.18 (t, 2H, J = 6.5 Hz,
COOCH2(CH2)4CH2O), 5.76 (t, 1H, J = 7.2 Hz, C1–H), 6.64 (s, 1H, C5–H,), 6.78 (s,
1H, C8–H), 6.80–8.06 (m, 10H, Ph-H); IR (KBr): 3134, 3019, 1641, 1610, 1551,
Compound 7g: 1H NMR (CDCl3, 300 MHz,
d
ppm): 2.72(t, 2H, J = 6.5 Hz,
1402 cmÀ1
36H41N3O11S: C, 59.74; H, 5.71; N, 5.81. Found: C, 59.77; H, 5.75; N, 5.78.
Compound 7p: 1H NMR (CDCl3, 300 MHz,
ppm): 1.53–2.36 (m, 8H,
;
MS (ESI, m/z): 746 ([M+ Na]+, base peak); Anal.Calcd. for
NOCCH2CH2COO), 2.80 (t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.89 (t, 2H,
J = 3.6 Hz, C4–H), 2.94–3.32 (m, 2H, C3–H), 3.85 (s, 6H, 2ÂOCH3), 4.14 (d, 2H,
J = 7.2 Hz, PhOCH2), 4.53 (t, 2H, J = 6.4 Hz, COOCH2CH2O), 4.63 (t, 2H, J = 6.4 Hz,
COOCH2CH2O), 5.72 (t, 1H, J = 7.2 Hz, C1–H), 6.63 (s, 1H, C5–H,), 6.73 (s, 1H, C8–
H), 6.85–8.07 (m, 10H, Ph-H); IR (KBr): 2926, 2851, 1733, 1626, 1576,
C
d
OCH2(CH2)4CH2O), 2.72 (t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.76 (t, 2H,
J = 6.5 Hz, NOCCH2CH2COO), 2.82 (t, 2H, J = 3.5 Hz, C4–H), 3.23–3.51 (m, 2H,
C3–H), 3.63–3.68 (d. 2H, J = 7.5 Hz, ArCH2), 3.75 (s, 3H, OCH3), 3.82 (s, 3H,
OCH3), 4.11 (t, 2H, J = 6.4 Hz, COOCH2(CH2)4CH2O), 4.14 (t, 2H, J = 6.5 Hz,
COOCH2(CH2)4CH2O), 5.75 (t, 1H, J = 7.5 Hz, C1–H), 6.36 (s, 1H, C5–H), 6.39 (s,
1H, C8–H), 7.64–8.17 (m, 12H, Ph-H); IR (KBr): 2937,2860, 1736, 1639, 1582,
1556, 1513, 1451 cmÀ1; MS (ESI, m/z): 780.2 ([M+ Na]+, base peak); Anal.Calcd.
for C40H43N3O10S: C, 63.39; H, 5.72; N, 5.54. Found: C, 63.37; H, 5.73; N 5.53.
17. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
;
1445 cmÀ1 MS (ESI, m/z): 668.2 ([M+H]+, base peak); Anal.Calcd. for
C
32H33N3O11S: C, 57.56; H, 4.98; N, 6.29. Found: C, 57.59; H, 5.01; N 6.27.
Compound 7h: 1H NMR (CDCl3, 300 MHz,
d
ppm): 2.76(t, 2H, J = 6.5 Hz,
NOCCH2CH2COO), 2.84 (t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 3.15 (t, 2H,
J = 3.6 Hz, C4–H), 3.67–3.71 (m, 2H, C3–H), 3.73 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3), 3.87 (d. 2H, J = 7.4 Hz, ArCH2), 4.55 (t, 2H, J = 6.4 Hz, COOCH2CH2O), 4.65
(t, 2H, J = 6.4 Hz, COOCH2CH2O), 5.75 (t, 1H, J = 7.4 Hz, C1–H), 6.48 (s, 1H, C5–H),
6.50 (s, 1H, C8–H), 7.60–8.09 (m, 12H, Ph-H); IR (KBr): 2931, 2844, 1740, 1622,
1545, 1514, 1450 cmÀ1; MS (ESI, m/z): 724.2 ([M+Na]+, base peak); Anal.Calcd.
for C36H35N3O10S: C, 61.62; H, 5.03; N, 5.99. Found: C, 61.57; H, 5.06; N, 5.96.
18. Cells were provided by the Institute of Hematology of Chinese Academy of
Medical Sciences. To perform growth experiments, Cells were grown in RPMI
1640 containing 10% fetal calf serum at 37 °C in
a 5% CO2 humidified
atmosphere. For the log phase cells were implanted in 96-well plates in the
density of 1 Â 105/mL, the original medium was removed and the cells were
incubated in the 180 mL RPMI 1640 containing no adriamycin for 14 days.
With different levels concentration various compounds (compounds 7a–p) or
the same volume PBS as vehicle control were added to incubate for 48 h.
During the last 4 h incubation the cells were determined by MTT assay. The
cells were exposed to MTT (5 mg/mL, sigma) and the resulting formazan
crystals were dissolved in 200 lM DMSO, followed by measuring at k = 570 nm
on a microplate reader (Thermo, USA). Assays were performed in duplicate,
with at least three separate experiments.
Compound 7i: 1H NMR (CDCl3, 300 MHz,
d ppm): 1.82–1.96 (m, 4H,
OCH2(CH2)2CH2O), 2.72(t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.75 (t, 2H,
J = 6.5 Hz, NOCCH2CH2COO), 2.83 (t, 2H, J = 3.4 Hz, C4–H), 3.69 (t, 2H,
J = 3.4 Hz, C3–H), 3.85 (s, 6H, 2ÂOCH3), 4.19 (t, 2H, J = 6.5 Hz,
COOCH2(CH2)2CH2O), 4.43 (t, 2H, J = 6.6 Hz, COOCH2(CH2)2CH2O), 4.64 (s, 2H,
C1–H), 6.59 (s, 1H, C5–H), 6.62(s, 1H, C8–H), 7.60–8.09 (m, 5H, Ph-H); IR (KBr):
2945, 2850,1733, 1615, 1552, 1515, 1455 cmÀ1; MS (ESI, m/z): 590.2 ([M+H]+,
base peak); Anal.Calcd. for C27H31N3O10S: C, 55.00; H, 5.30; N, 7.13. Found: C,
54.97; H, 5.26; N, 7.13.
Compound 7j: 1H NMR (CDCl3, 300 MHz,
d
ppm): 1.80–1.96 (m, 4H,
19. MDR reversal activity was reported as IC50 values in the K562/A02 cell line.
Cells were seeded into 96-well plates at 3 Â 104 cells/well. Various
concentrations of adriamycin and the tested compounds were gradually
added and plates were incubated in 5% humidified incubator at 37 °C for 48 h.
Finally, the MTT assay was performed as mentioned before. IC50 values of
adriamycin (concentration resulting in 50% inhibition of cell growth) were
calculated in the presence of five concentrations of the tested compounds (0.1,
1.0, 10, 20, and 50 lM).
20. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer
Res. 1987, 47, 936.
21. Jabbar, S. A.; Twentyman, P. R.; Watson, J. V. Br. J. Cancer 1989, 60, 523.
22. Lambert, E.; Rees, J. K.; Twentyman, P. R. Leukemia 1992, 6, 1063.
23. Lella, J. W. Bull. Hist. Med. 2001, 75, 760.
24. K562/A02 cells were grown in RPMI 1640 containing 10% fetal calf serum at
37 °C in a 5% CO2 humidified atmosphere. For the log phase, cells were
implanted in 96-well plates in the density of 1 Â 106/mL, the original medium
was removed and the cells were incubated in the 180 mL RPMI 1640
OCH2(CH2)2CH2O), 2.71(t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.72 (t, 2H,
J = 6.5 Hz, NOCCH2CH2COO), 2.73–2.98 (m, 2H, C4–H), 3.06(t, 2H, J = 7.5 Hz,
ArCH2), 3.41–3.44(m, 1H, C3–H), 3.68–3.70 (m, 1H, C3–H), 3.83 (s, 6H, 2ÂOCH3),
3.86 (s, 6H, 2ÂOCH3), 4.13 (t, 2H, J = 6.4 Hz, COOCH2(CH2)2CH2O), 4.43 (t, 2H,
J = 6.6 Hz, COOCH2(CH2)2CH2O), 5.56 (t, 1H, J = 7.5 Hz, C1–H), 6.17–6.20 (m, 3H,
Ar-H), 6.61 (s, 1H, C5–H), 6.62 (s, 1H, C8–H), 7.60–8.08 (m, 5H, Ph-H); IR (KBr):
2927, 2844, 1732, 1625, 1554, 1514, 1449 cmÀ1; MS (ESI, m/z): 740.2 ([M+H]+,
base peak); Anal.Calcd. for C36H41N3O12S: C, 58.45; H, 5.59; N, 5.68. Found: C,
58.44; H, 5.58; N, 5.71.
Compound 7k: 1H NMR (CDCl3, 300 MHz,
d
ppm): 1.81–2.17 (m, 4H,
OCH2(CH2)2CH2O), 2.70(t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.78 (t, 2H,
J = 6.5 Hz, NOCCH2CH2COO), 2.90 (t, 2H, J = 3.4 Hz, C4–H), 2.93–3.25 (m, 2H,
C3–H), 3.81 (d, 2H, J = 7.5 Hz, PhOCH2), 3.83 (s, 3H, OCH3), 3.88(s, 3H, OCH3),
4.17 (t, 2H, J = 6.4 Hz, COOCH2(CH2)2CH2O), 4.44 (t, 2H, J = 6.5 Hz,
COOCH2(CH2)2CH2O), 5.75 (t, 1H, J = 7.5 Hz, C1–H), 6.63 (s, 1H, C5–H,), 6.73 (s,
1H, C8–H), 6.85–8.07 (m, 10H, Ph-H); IR (KBr): 2935, 2837, 1732, 1643, 1601,
1586, 1552, 1517 cmÀ1; MS (ESI, m/z): 696.2 ([M+H]+, base peak); Anal.Calcd.
for C34H37N3O11S: C, 58.70; H, 5.36; N, 6.04. Found: C, 58.73; H, 5.35; N, 5.40.
containing no adriamycin for 14 days. With 5
lg/mL rhodamine123 in the
absence and presence of 10 M 7d, 7f, 7l or verapamil were incubated for
l
Compound 7l: 1H NMR (CDCl3, 300 MHz,
d
ppm): 1.81–2.16 (m, 4H,
60 min at 37 °C in a 5% CO2 humidified atmosphere. After incubation, the cells
were centrifugeed and washed twice with ice-cold phosphate-buffered saline,
resuspended in 200 mL PBS and then analysed by flow cytometry (FACS
Callibur: BD) (lEx = 488 nm, lEm = 570 nm), and the concentration of
OCH2(CH2)2CH2O), 2.73(t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.80 (t, 2H,
J = 6.5 Hz, NOCCH2CH2COO), 3.18 (t, 2H, J = 3.6 Hz, C4–H), 3.68–3.72 (m, 2H,
C3–H), 3.81 (d, 2H, J = 7.5 Hz, ArCH2), 3.84 (s, 6H, 2ÂOCH3), 4.21 (t, 2H,
J = 6.5 Hz, COOCH2(CH2)2CH2O), 4.45 (t, 2H, J = 6.6 Hz, COOCH2(CH2)2CH2O),
5.55 (t, 1H, J = 7.5 Hz, C1–H), 6.39 (s, 1H, C5–H), 6.42 (s, 1H, C8–H), 7.60–8.06
(m, 12H, Ph-H); IR (KBr): 2933, 2873, 1736, 1608, 1590, 1514, 1455 cmÀ1; MS
(ESI, m/z): 752.2([M+Na]+, base peak); Anal.Calcd. for C38H39N3O10S: C, 62.54;
H, 5.39; N, 5.76. Found: C, 62.54; H, 5.43; N, 5.78.
rhodamine123 was measured from the fluorescence value by using
a
rhodamine123 standard curve. The fluorescence value was regarded as
relative concentration of rhodamine123 in the cells.
25. Ludescher, C.; Thaler, J.; Drach, D.; Drach, J.; Spitaler, M.; Gattringer, C.; Huber,
H.; Hofmann, J. Br. J. Haematol. 1992, 82, 161.
26. Fontaine, M.; Elmquist, W. F.; Miller, D. W. Life Sci. 1996, 59, 1521.
27. The levels of nitrate/nitrite formed from individual compounds in the cells
were determined by the colorimetric assay using the nitrate/nitrite
colorimetric assay kit, according to the manufacturer’s instructions. Briefly,
Compound 7m: 1H NMR (CDCl3, 300 MHz,
d
ppm): 1.60–1.89 (m, 8H,
OCH2(CH2)4CH2O), 2.68(t, 2H, J = 6.5 Hz, NOCCH2CH2COO), 2.75 (t, 2H,
J = 6.5 Hz, NOCCH2CH2COO), 2.83 (t, 2H, J = 3.3 Hz, C4–H), 3.69 (t, 2H,
J = 3.3 Hz, C3–H), 3.85 (s, 6H, 2ÂOCH3), 4.09 (t, 2H, J = 6.5 Hz,
COOCH2(CH2)4CH2O), 4.14 (t, 2H, J = 6.5 Hz, COOCH2(CH2)4CH2O), 5.30 (s, 2H,
C1–H), 6.59 (s, 1H, C5–H), 6.62 (s, 1H, C8–H), 7.59–8.07 (m, 5H, Ph-H); IR (KBr):
2936, 2859, 1745, 1710, 1645, 1620, 1549, 1517, 1454 cmÀ1; MS (ESI, m/z):
618.3 ([M+H]+, base peak); Anal.Calcd. for C29H35N3O10S: C, 56.39; H, 5.71; N,
6.80. Found: C, 56.38; H, 5.68; N, 6.77.
K562/A02 cells (5 Â 106 per well) were treated in triplicate with 10
lM of one
of the compounds (7d, 7f, 7l, and HZ08) for 24 h, respectively. The cells were
harvested lysed. The cell lysates were mixed with Griess for 30–300 min,
followed by measuring at 570 nm. The cells treated with diluent (control
group) were used as negative controls for the background levels of nitrate/
nitrite production, while with sodium nitrate at different concentrations was
used as positive controls for the standard curve.
Compound 7n: 1H NMR (CDCl3, 300 MHz,
d ppm): 1.56–2.04 (m, 8H,
OCH2(CH2)4CH2O), 2.62 (t, 2H, J = 6.7 Hz, NOCCH2CH2COO), 2.69 (t, 2H,
J = 6.7 Hz, NOCCH2CH2COO), 2.70–2.98 (m, 2H, C4–H), 3.10 (d, 2H, J = 7.5 Hz,