Design and synthesis of tetrahydroisoquinoline derivatives as potential multidrug resistance reversal agents in cancer

Article abstract of DOI:10.1016/j.bmcl.2008.04.082

Exploration for new MDR-modulator utilizing tetrahydroisoquinoline as scaffold disclosed 6,7-dimethoxy-1-(3,4-dimethoxy)benzyl-2-(N-n-octyl-N′-cyano)guanyl-1,2,3,4-tetrahydroisoquinoline (7) as a readily accessible medicinal lead. Compound 7 possessed potent MDR reversal activity in the range of the reference compound verapamil, and had not cardiovascular activity compared to verapamil. Crown Copyright

Full text of DOI:10.1016/j.bmcl.2008.04.082

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3652–3655
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of tetrahydroisoquinoline derivatives as potential
multidrug resistance reversal agents in cancer
a,
b
Yu Li a, Hui-bin Zhang a, , Wen-long Huang , Yun-man Li
*
*
a Center of Drug Discovery, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China
b 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:
Exploration for new MDR-modulator utilizing tetrahydroisoquinoline as scaffold disclosed 6,7-dime-
thoxy-1-(3,4-dimethoxy)benzyl-2-(N-n-octyl-N0-cyano)guanyl-1,2,3,4-tetrahydroisoquinoline (7) as
a
Received 6 January 2008
Revised 22 March 2008
Accepted 19 April 2008
Available online 11 May 2008
readily accessible medicinal lead. Compound 7 possessed potent MDR reversal activity in the range of
the reference compound verapamil, and had not cardiovascular activity compared to verapamil.
Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
Keywords:
Tetrahydroisoquinoline derivatives
Multidrug resistance
P-glycoprotein
Multidrug resistance (MDR)1 is a major problem in cancer treat-
ment. The typical MDR in tumor cells is mainly associated with a
reduced intracellular drug accumulation and an increased cellular
drug efflux. This phenomenon can be related to the overexpression
activities in vitro were evaluated, and the results showed that these
compounds exerteddifferentdegreesof MDRreversalactivities. Par-
ticularly, the activity of 6,7-dimethoxy-1-(3,4-dimethoxy)benzyl-
2-(N-n-octyl-N0-cyano)guanyl-1,2,3,4-tetrahydro-isoquinoline (7)
was comparable to that of the control verapamil.13
of the energy-dependent efflux pump, P-glycoprotein (P-gp),2
a
170-kDa protein that belongs to the ATP-binding cassette super-
family of transporters. Intense efforts to overcome MDR by influ-
encing transporter expressions via signal transduction pathways
or by direct transcriptional control have not been successful in
clinical trials.3 A number of compounds, so-called chemosensitiz-
ers, are able to reverse the effect of Pgp on MDR.4 Tsuruo and co-
workers5 were the first to demonstrate the ability of the calcium
channel blocker, verapamil, to reverse MDR. The cardiovascular ac-
tion of verapamil derivatives has always represented a problem in
the development of MDR modulators possessing this structure and
many efforts have been devoted to identifying more selective
compounds.
Several bisbenzylisoquinoline alkaloids as tetrandrine and berb-
amine show anti-MDR properties and calcium antagonistic activity
in various degrees.6 More than 100 tetrahydroisoquinoline deriva-
tives were designed and synthesized for the search of novel
calcium channel blockers by simplifying and optimizing tetrandrine
in our group.7–12 A series of N-cyanoguanyl-substituted tetrahydro-
isoquinoline derivatives had strong calcium antagonistic activities
but showed almost no cardiovascular activities. Their MDR reversal
In this letter, the representative compound 7 was synthesized
and evaluated in several assays of MDR reversal and cardiovascular
activities in vitro and in vivo.
The synthetic route to the target compound 7 is outlined in
Scheme 1. The synthesis of compound 7 utilized 3,4-dimethoxy-
phenylethyamine 1 and 3,4-dimethoxy-phenylacetic acid 2 as the
starting material, which were converted to3 at 190 °C under N2
protection in 84% yield. Treatment of 3 with POCl3 in toluene under
reflux provided 4 in 97% yield, followed by reduction with the yield
of 64%. Next, the reaction at the N-position of compound 5 was
achieved with dimethyl cyanocarbonimidodithioate in 25% yield.
Reaction of key intermediate 6 with n-octylamine in toluene under
reflux gave 7 with the yield of 34%. Compound 7 was characterized
by IR, 1HNMR, mass spectra, and elemental analysis.14
The in vitro MDR reversal activities of compound 7 against
MCF-7, MCF-7/ADR, and K562/A02 cell lines were evaluated by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT) colorimetric assay15 with verapamil as reference drug
(Tables 1 and 2). The results showed that compound 7 exhibited a
well-defined trend in MDR reversal activities.
The in vivo efficacy of compound 7 was evaluated by using the
resistant K562/A02 cell xenografts SCID nude mice.16 The results
displayed that compound 7 had no direct effect on K562/A02 cell
growth. The antitumor activity of adriamycin (ADM) was signifi-
* Corresponding authors.
E-mail addresses: zhanghb80@163.com (H. Zhang), ydhuangwenlong@126.com
(W. Huang).
0960-894X/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.082

Y. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3652–3655
3653
H
N
H3CO
H3CO
OCH3
OCH3
H3CO
H3CO
H3CO
H3CO
NH2
COOH
a
+
O
1
2
3
H3CO
H3CO
H3CO
H3CO
b
c
d
N
NH
OCH3
OCH3
OCH3
OCH3
4
5
CN
N
CN
N
H3CO
H3CO
H3CO
H3CO
e
N
SMe
N
NHC8H17-n
OCH3
OCH3
OCH3
OCH3
6
7
Scheme 1. Reagents and conditions: (a) N2, 190 °C, 3 h; (b) POCl3, toluene, N2, 110 °C, reflux, 2.5 h; (c) KBH4, diethylamine, methanol, rt, 22 h; (d) dimethyl cyanocarbon-
imidodithioate, toluene, reflux, 30–50 h; (e) n-C8H17NH2, toluene, reflux, 14–72 h.
Table 1
Inhibitory effects of compound 7 on the proliferation of MCF-7 cell line and MCF-7/
ADR cell line by MTT assay(X ꢀ s; n ¼ 5)
Fluorescence value
ꢀ
4500
4000
3500
3000
2500
2000
1500
1000
500
10µM
*
*
Compound#
IC50 MCF-7
MCF-7##
IC50 MCF-/ADR
MCF-7/ADR##
98.67%
69. 29%
3µM
1µM
35.18%
Control**
Verapamil
7
96.2 2.6
92.8 1.8
79.7 0.9
6864.2 4.9
689.2 8.6
226.8 3.5
60.47%
1.0
1.2
10.0
30.3
*
IC50 of adriamycin (nmol/L).
**
#
0.01‰ DMSO.
Compounds were tested at 10 lmol/L.
##
Reversal fold.
0
Table 2
7
Control
Verapamil
Inhibitory effects of compound 7 on the proliferation of K562/A02 cell line by MTT
ꢀ
assay(X ꢀ s; n ¼ 5)
Figure 1. The effects of compound 7 on cellular Rh123 accumulation in MCF-7/
ADM (n = 3).
Compound#
Reversal adriamycin
Reversal vincristine
*
##
*
##
IC50
IC50
Control**
Verapamil
7
18.13 1.2
0.40 0.2
0.38 0.3
12.23 0.4
1.55 0.1
1.33 0.1
cantly potentiated by the coadminstration of compound 7 (8, 4 and
2 mg/kg) in SCID nude mice (Table 3).
45.30
47.71
7.90
9.20
The rhodamine 123 (Rh123) accumulation assay was used to
measure the P-gp inhibitory activity of compound 7. The uptake
of Rh123 in cells was followed by monitoring the fluorescence sig-
nal with the method described.17 The results showed that fluores-
cence value of compound 7 was increased obviously compared to
*
IC50 of adriamycin (lmol/L).
0.01‰ DMSO.
Compounds were tested at 10 lmol/L.
**
#
##
Reversal fold.
Celluar Rh123
(µmol/g protein)
1.4
Table 3
10µM
**
10µM
The effects of compound
7 on K562/A02 cell growing of bearing cancer mice
3µM **
ꢀ
(X ꢀ s; n ¼ 8)
*
1µM
*
1.2
1
Group
Dose (mg/kg)
Tumor weight (g)
Inhibitory ratio (%)
0.9% NaCl
0.9% NaCl + ADM
Verapamil
Verapamil + 7
ADM
—
—
8
8
2
8
2
8
4
2
3.54 1.1*
2.15 0.4#
3.61 1.2*
ꢁ63.1
0.9
ꢁ66.4
0.8
0.6
0.4
0.2
0
0.3 0.2**,##
2.17 0.4#
3.46 1.2*
86.2
7
ꢁ59.5
ꢁ62.2
85.3
81.6
70.5
3.52 1.1*
7 + ADM
0.32 0.2**,##
0.4 0.3**,##
0.64 0.3**,##
*
Control
7
Verapamil
P < 0.05.
P < 0.01 vs ADM.
P < 0.05.
P < 0.01 vs 0.9%NaCl.
**
#
Figure 2. The effects of compound 7 on Rh123 accumulation in RBMEC (*P < 0.05,
**P < 0.01 vs control) (n = 3).
##

3654
Y. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3652–3655
Table 5
control. P-gp function in MCF-7/ADM cells was inhibited after the
treatment with compound 7. When compound 7 was added at a
concentration of 1, 3 and 10 lmol/L, the inhibitory ratio was
35.18%, 60.47%, and 69.29%, respectively (Fig. 1).
ꢀ
Antiarrhythmic action of compound 7 by aconitine nitrate (X ꢀ s; n ¼ 3)
Group
Dose (mg/kg)
The dose of aconitine nitrate when antiarrhythmic
(lg/kg)
P-gp, which actively transport agents from the cerebral endo-
thelial cells back into the bloodstream, have a major influence on
blood–brain barrier (BBB) permeability to hydrophobic and amphi-
philic drugs.18 In order to further investigate the effect of com-
pound 7 on P-gp, the rat brain microvascular endothelial cells
(RBMEC)19–21 for BBB model have been used. After incubated with
RBMEC, compound 7 of 1, 3, and 10 lmol/L could increase the
intracellular accumulation of Rh123 significantly22 (Fig. 2).
The increase in the efflux of drugs is the major feature of P-gp
mediated MDR. Considering that Rh123 efflux is more sensitive
than that of anticancer drugs, the efflux inhibitory activity of the
compound with Rh123 as a substrate was chosen to quantify the
MDR reversal activity.23 This test was carried out on RBMEC cell
line. A time-dependent Rh123 content curve was performed with
verapamil as reference drug (Fig. 3). The results showed that
Rh123 efflux was decreased in the dose-dependent manner.
In parallel with the mechanisms of MDR modulation it could be
speculated that the ability of the drugs to interact with the mem-
VP
VT
VF
Control
7
Verapamil
20.2 0.1
22.4 0.1
30.3 0.2
28.9 0.3
26.7 0.1
44.0 0.2
37.0 0.2
31.5 0.1
55.0 0.1
2
2
brane phospholipids could also play a role.24 Drug/membrane
interactions could lead directly (changes in membrane permeabil-
ity and fluidity) and/or indirectly (inhibition of P-gp phosphoryla-
tion via inhibition of PKC, changes of the conformation and
functioning of the membrane-integrated proteins via changes in
the structure organization of the surrounding membrane bilayer)
to the reversal of MDR.25 Diphenylhexatriene(DPH) was chosen
to quantify the membrane lipid fluidity.26 The results showed that
the membrane lipid fluidity in K562 cell line was not affected by
compound 7 while that in K562/A02 cell line was decreased after
incubated with 10 lmol/L compound 7 (P < 0.05) (Fig. 4). The re-
sults indicated that the mechanism of compound 7 may inhibit
P-gp transport function through decreasing membrane lipid fluid-
ity indirectly.
The in vitro vascular contractile activity of compound 7 were
performed with aorta isolated from SD rats (250–350 g).27 Verapa-
mil was used as reference drug. The results showed that compound
7 had no vascular contractile activity (Table 4).
The in vivo arrhythmogenesis was evaluated following the
method described.28 The dose of aconitine nitrate was determined
after drug injection by echocardiography when ventricular prema-
ture beat (VP), sustained tachycardia (VT), and fibrillation (VF)
happened. The results showed that compound 7 had lower
arrhythmogenic effect than verapamil (Table 5).
In summary, tetrahydroisoquinoline derivative 7 was synthe-
sized and evaluated for its MDR reversal and cardiovascular activ-
ities in vitro and in vivo. Compound 7 might exhibit MDR reversal
activity by directly modulating the function of P-gp or indirectly
inhibition of P-gp transport function through decreasing mem-
brane lipid fluidity. Compound 7 may be a promising MDR reversal
drug candidate. Further biological evaluation, pharmacokinetics
study, long-term toxicity, and mechanistic studies on this new
compound are currently in progress and will be reported in due
course.
Cellular Rh123
Control
(µmol/g protein)
Verapamil
6
1µM 7
5
3µM 7
10µM7
4
3
2
1
0
*
*
**
*
*
**
*
5
15
30
60
90
Time(min)
Figure 3. The effects of compound 7 on Rh123 efflux in RBMEC (*P < 0.05, **P < 0.01
vs control) (n = 3).
Fluorescence polarization
10µM
(P)
*
0.192
0.19
3µM
0.188
0.186
0.184
0.182
0.18
Acknowledgment
10µM
The work was supported by the Hi-tech Research and Develop-
ment Program of China (No.2002AA233071).
1µM
References and notes
0.178
0.176
0.174
1. Biedler, J. L.; Chang, T.; Meyers, M. B.; Peterson, F. H.; Spengler, B. A. Cancer
Treatment Rep 1983, 67, 859.
2. Cole, S. P. C.; Deeley, R. G. Bioassays 1998, 20, 931.
Control
Verapamil
7
3. Dassow, H.; Lassner, D.; Remke, H.; Preiss, R. Int. J. Clin. Ther. 2000, 38, 209.
4. Teodori, E.; Dei, S.; Scapecchi, S.; Gualtieri, F. Farmaco 2002, 57, 385.
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03113262.6.
Figure 4. The effects of compound 7 on membrane lipid fluidity in K562/A02 (*P -
< 0.05 vs control).
Table 4
ꢀ
The inhibitory ratio of compound 7 on aorta contractile activity (X ꢀ s; n ¼ 3)
Group
Concentration
20 mmol KCl
80 mmol KCl
Verapamil
7
10ꢁ6 mol/L
10ꢁ6 mol/L
36.3 0.2%
46.3 0.1%
14. Selected spectroscopic data: Compound 7: IR (KBr, m): 3380, 3000, 2920, 2860,
0
0.0%
0
0.0%
2162, 1610, 1570, 1520 cmꢁ1 1H NMR (CDCl3, 300 MHz, d ppm): 6.40–6.84 (m,
;

Y. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3652–3655
3655
5H, Ar–H), 4.95–5.03 (m, 1H, C1–H), 3.78 (s, 3H, OCH3), 3.86 (s, 9H, 3ꢂ OCH3),
2.77–3.66 (m, 8H, C3–H, C4–H, ArCH2, NHCH2), 0.88–1.62 (m, 15H, (CH2)6CH3);
MS(ESI, m/z): 523 ([M+H]+, base peak); Anal. Calcd for C30H42N4O4: C 68.94, H
8.10, N 10.72; found: C 68.58, H 8.13, N 10.65.
atmosphere. Primary culture RBMEC cells were cultured in 24-well plates.
When cells were forming a 90% confluent monolayer, the original medium was
removed and the cells were incubated in the 0.45-ml assay buffer (PH 7.4) and
0.05-ml Rh123 with 100 lmol/L for 30 min. After being washed three times
with ice-cold PBS, the samples were resuspended in assay buffer (PH 7.4) with
Rh123-free. Verapamil with 10 lmol/L as a final concentration and 10, 3, and
1 lmol/L compound 7 were added to incubate. The cells were centrifuged after
adding ice-cold PBS in different time and washed three times with ice-cold
PBS. Finally, 1% of Trition X-100 cells were added to each well. The intracellular
fluorescence due to Rh123 (kexcitation = 488 nm, kemission = 535 nm) was
measured in a microplate spectrofluorometer. The content of protein was
measured using the method of Coomassie brilliant blue for fluorescence
intensity value. Assays were performed in duplicate, with at least three
separate experiments.
15. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
16. K562/A02 cells were grown in RPMI 1640-containing 10% fetal calf serum and
adriamycin (ADM) with 1 lg/ml as a final concentration at 37 °C in a 5% CO2
humidified atmosphere. For the log phase cells were inoculated
subcutaneously in oxter in the density of 1 ꢂ 107/ml. When the tumors
reached a mean diameter of 0.4–0.6 cm, the animals were randomized into
groups of ten and treated with drugs in the intravenous injection three times a
week. Control animals were treated with vehicle alone. Tumor weights were
measured. The inhibition ratio (I%) of tumor growth was calculated using
((mean relative tumor weights of the treated group)/(the mean relative tumor
weights of ADM group) ꢁ 1) ꢂ 100 (%).
24. Seydel, J. K.; Velasco, M. A.; Coats, E. A.; Cordes, H. P.; Kunz, B.; Wiese, M. Quant.
Struct.-Act. Relat. 1992, 11, 205.
25. Pajeva, I. K.; Wiese, M.; Cordes, H. P.; Seydel, J. K. J. Cancer Res. Clin. Oncol. 1996,
122, 27.
17. MCF-7/ADM cells were grown in RPMI 1640-containing 10% fetal calf serum,
ADM with 1 lmol/L as a final concentration at 37 °C in a 5% CO2 humidified
atmosphere. For the log phase cells were implanted in 96-well plates in the
density of 4 ꢂ 104/ml. the original medium was removed and the cells were
incubated in the 160 lL RPMI 1640 containing no serum for 30 min. Verapamil
with 10 lmol/L as a final concentration and 10, 3 and 1 lmol/L compound 7
and the same volume of PBS were added to incubate for 30 min. Then
26. K562 and 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 ꢂ 105/ml. A final concentration
of 10, 3, and 1 lmol/L compound 7 was added, respectively. Ten micromoles
per liter verapamil and PBS was added as reference. After 24 h of incubation.
The cells were centrifuged and washed twice with ice-cold PBS, the samples
were resuspended in 1 ml PBS. DPH buffer (1ml) with 2 lmol/L were added for
30 min. After being washed twice with ice-cold PBS, the samples were
resuspended in 2 ml PBS and incubated for 10 min at 25 °C. Four direction of
fluorescence polarization were measured by adding two polarizing filters
vertically (kexcitation = 357.6 nm, bandpass = 10 nm, kemission = 428.7 nm) in a
microplate spectro-fluorometer immediately. To calculate the Fluorescence
polarization (P) for expressing the membrane lipid fluidity (P relating with
membrane fluidity in inverse ratio) by the equation: P = (IVV ꢁ G ꢂ IVH)/
(IVV + G ꢂ IVH); where IVH and IVV are fluorescence intensity value when
excitation in vertical direction, G is correction factor (G = IHV/IHH), where IHV
and IHH are fluorescence intensity value when excitation in horizontal
direction. Assays were performed in duplicate, with at least three separate
experiments.
rhodamine 123 with 10 lmol/L as
a final concentration was added and
incubation continued for 60 min at 37 °C. After being washed twice with
phosphate-buffered saline (PBS), 1% of Trition X-100 cells were added to each
well. The samples were resuspended in PBS for analysis. The intracellular
fluorescence due to Rh123 (kexcitation = 488 nm, kemission = 535 nm) was
measured in
a microplate spectrofluorometer. Assays were performed in
triplication. The inhibitory ratio (I%) was calculated using ((mean absolute
fluorescence value of the treated group ꢁ the mean absolute fluorescence
value of control)/the mean absolute fluorescence value of control) ꢂ 100 (%),
where absolute fluorescence value for (fluorescence value of each well) ꢁ (the
bases of fluorescence value of 96-well plate).
18. Schinkel, A. H.; Smit, J. J. M.; Tellingem, O.; Beijnen, J. H.; Wagenaar, R.; Demter,
L.; Mol, C. A. A. M.; Valk, M. A.; Maandag-Robanu, E. C.; Riele, H. P. J.; Berns, A. J.
M.; Borst, P. Cell 1994, 77, 491.
19. Bowman, P. D.; Betz, A. L. In Vitro 1998, 34, 777.
20. Zhang, L.; Liu, X. D.; Xie, L. Acta Pharmacol. Sinica 2003, 24, 903.
21. Xu, Y. G.; Liu, Y. J. Chin. Microcirculation 1997, 2, 63.
22. RBMEC cells were grown in DF containing 20% fetal calf serum at 37 °C in a
5% CO2 humidified atmosphere. Primary culture RBMEC cells were cultured
in 24-well plates. When cells were forming a 90% confluent monolayer, the
original medium was removed and the cells were incubated in the 0.4 ml DF
27. A section of the aorta was cleared of adhering fat and connective tissue and
was cut into transverse rings (4- to 5-mm-long). The endothelium was
removed by rubbing the intimal surface with forceps. The segments were
suspended under 1.0 g of tension by means of steel hooks in an organ bath
containing 10 mL of a Krebs-bicarbonate-buffered solution of the following
composition (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, NaHCO3 25, KH2PO4 1.2,
MgSO4 1.2, glucose 5. The physiological solutions were maintained at 37 °C and
bubbled continuously with a mixture of O2 (95%) and CO2 (5%). The isometric
contractions of the aortic rings were measured with a force–displacement
transducer. After 60 min of equilibration, the rings were exposed to 20 mM KCl
or 80 mM KCl. When the tension had stabilized, the drugs were added to the
bath at increasing concentrations until maximal relaxation. The relaxation
response was expressed as the percentage of the contractile response to
KCl.
containing no serum for 30 min. Verapamil with 10 lmol/L as
a final
concentration and 10, 3, and 1 lmol/L compound 7 were added, the same
volume of PBS and Rh123 with 5 lmol/L as a final concentration in each
well was added at last to incubate for 90 min. The cells were centrifuged
after adding ice-cold PBS in different time and washed three times with ice-
cold PBS. Finally, 1% of Trition X-100 cells were added to each well. The
intracellular
fluorescence
due
to
Rh123
(kexcitation = 488 nm,
kemission = 535 nm) was measured in
a
microplate spectrofluorometer. The
content of protein was measured using the method of Coomassie brilliant
blue for fluorescence intensity value. Assays were performed in duplicate,
with at least three separate experiments.
28. SD rats (2.5–3.5 kg) were anesthetized with urethane (1.2 g/kg ip). The test
compounds were dissolved in normal saline and administrated by femoral
vein. Intravenous administration of aconitine nitrate (1 lg/0.2 ml/min)
was kept constant velocity from femoral vein by peristaltic pump after
2 min.
23. Compound 7 was diluted in DMSO (10 mmol/L) and RBMEC cells were grown
in DF containing 20% fetal calf serum at 37 °C in
a 5% CO2 humidified