Synthesis and biological evaluation of taxinine analogues as orally active multidrug resistance reversal agents in cancer

Article abstract of DOI:10.1016/j.bmcl.2004.06.089

Three novel taxinine analogues were prepared and tested for their activity as multidrug resistance (MDR) reversal agents in comparison with verapamil. In vitro testing demonstrated that compounds 8-10 possess MDR-reversal activity in the KB/V cell line. In accumulation assay, the intracellular rhodamine123 concentration increased significantly after treatment with compound 9, higher than that of verapamil in KB/V cell line. In vivo studies with VCR-resistant KB/V tumor xenografts showed that 9 in combination with VCR significantly inhibited tumor growth. Treatment with VCR or 9 alone did not result in growth inhibition. Three novel taxinine analogues were prepared and tested for their activity as multidrug resistance (MDR) reversal agents in comparison with verapamil. In vitro testing demonstrated that compounds 8-10 possess MDR-reversal activity in the KB/V cell line. Half-hour after treatment with 5, 10, and 20 μmol/L compound 9, the intracellular rhodamine123 concentration increased 2.3, 2.9, and 3.2-fold, respectively, higher than 1.88-fold of 10 μmol/L verapamil in KB/V cell line. In vivo studies with VCR-resistant KB/V tumor xenografts showed that compound 9 in combination with VCR significantly inhibited tumor growth. Treatment with VCR or 9 alone did not result in growth inhibition. These results reveal that three taxinine analogues are good modifiers of MDR in tumor cells.

Full text of DOI:10.1016/j.bmcl.2004.06.089

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4767–4770
Synthesis and biological evaluation of taxinine analogues as
orally active multidrug resistance reversal agents in cancer
Xin Zhao,a, Jun Gu,b, Dali Yinb and Xiaoguang Chena,
*
aDepartment of Pharmacology, Institute of Materia Medica, Chinese Academic of Medical Science and Peking Union
Medical College, 1 Xian Nong Tan Street, Beijng 100050, China
bDepartment of Medicinal Chemistry, Institute of Materia Medica, Chinese Academic of Medical Science and Peking Union
Medical College, 1 Xian Nong Tan Street, Beijng 100050, China
Received 18 May 2004; accepted 21 June 2004
Available online 24 July 2004
Abstract—Three novel taxinine analogues were prepared and tested for their activity as multidrug resistance (MDR) reversal agents
in comparison with verapamil. In vitro testing demonstrated that compounds 8–10 possess MDR-reversal activity in the KB/V cell
line. Half-hour after treatment with 5, 10, and 20lmol/L compound 9, the intracellular rhodamine123 concentration increased 2.3,
2.9, and 3.2-fold, respectively, higher than 1.88-fold of 10lmol/L verapamil in KB/V cell line. In vivo studies with VCR-resistant
KB/V tumor xenografts showed that compound 9 in combination with VCR significantly inhibited tumor growth. Treatment with
VCR or 9 alone did not result in growth inhibition. These results reveal that three taxinine analogues are good modifiers of MDR in
tumor cells.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Multidrug resistance (MDR) in tumor cells is a signifi-
cant obstacle to the success of chemotherapy in many
cancers. MDR is a phenomenon whereby tumor cells
that have been exposed to one cytotoxic agent develop
cross-resistance to a range of structurally and function-
ally unrelated compounds. The drug resistance that
develops in cancer cells often results from elevated
expression of particular proteins, such as cell-membrane
transporters, which can result in an increased efflux of
the cytotoxic drugs from the cancer cells, thus lowering
their intracellular concentrations. The classic resistance
to the cytotoxic drugs has most often been linked to
the overexpression of P-glycoprotein (P-gp), a 170-kd
ATP-dependent membrane transporter that acts as a
drug efflux pump, resulting in increased efflux of chemo-
therapeutic agents from cancer cells.1 P-gptransports a
broad range of substrates. To inhibit P-gpas a method
to reverse MDR in cancer patients has been studied
extensively, some compounds have been developed to
reverse MDR in vitro and in vivo.2 Some nontaxol-type
taxoids, including taxinine (1) isolated from Japanese
yew Taxus cuspidate, have been reported as reversal
agents.3 Sinenxan A (SIA) (2) is readily available as a
biosynthetic taxane in good yield.4 It has similar skele-
ton with taxinine (1). The development of a procedure
using SIA (2) as starting material for the preparation
of bioactive taxinine analogues would be of significance
(Fig. 1).
In this paper we describe the preparation of three taxi-
nine analogues synthesized from SIA (2), the effects on
cellular accumulation of rhodamine123 (Rho.123) in
MDR tumor cells, and the reversal activity in tumor
xenografts.
The procedure for the synthesis of compounds 8–10
from SIA (2) is outlined in Scheme 1. The acetyl at
C-14 was removed by KOH/MeOH/THF at 0ꢁC to give
compound 3 in approximately 30% yield, accompanied
AcO
AcO
OAc
H
10
O
14
O
H
AcO
H
OAc
sinenxan A ( 2 )
Figure 1. The structures of taxinine (1) and sinenxan A (2).
H
O
OAc
OAc
*
Corresponding author. Tel.: +86-10-63165207; e-mail: anthony_
zhao@hotmail.com
These authors contributed equally to this work.
taxinine
( 1 )
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.089

4768
X. Zhao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4767–4770
AcO
AcO
AcO
c
a
b
S
O
H
H
H
AcO
H
HO
OAc
H
S
OAc
H
OAc
OAc
OAc
OAc
4
3
2
AcO
AcO
AcO
f
e
O
d
O
H
H
H
OH
H
OAc
H
H
OAc
OAc
OAc
OAc
6
7
5
AcO
R = PhCH2
8:
9:
O
R = PhCH2CH2
dimethoxyl-PhCH
R = 2,5-
10:
2
H
H
O
R
OAc
O
Scheme 1. Reagents and reaction conditions: (a) 10mol/L KOH, MeOH, THF, 0ꢁC, 20min, 30–40%; (b) NaH, CS2, dry THF, N2, reflux, 15h; then
MeI, 40ꢁC, 3h, 85% (two steps); (c) H3PO2, AIBN, dioxane, cyclohexene, reflux, 5h, 82%; (d) 70% t-BuOOH, CrO3, CH2Cl2, rt, 5h, 73%;
(e) t-BuOK, dry THF, N2, ꢀ78ꢁC, 2h, 90%; (f) RCOOH, DCC, DMAP, dry toluene, rt, 6h, 8 (85%), 9 (88%), 10 (83%).
by 10-OH-SIA and recovered SIA (2). The treatment of
compound 3 with CS2/NaH/MeI in THF gave xanthated
compound 4 in 85% yield. By radical deoxygenation and
allylic oxidation,5 13-keto derivative (6) could be ob-
tained. The 5-O-acetate of 6 could be regio-selectively
cleaved by t-BuOK in THF to give 7 in high yield.6
The target compounds 8, 9, 10 were obtained by esteri-
fication with the corresponding acid. The structures of
Table 1. Effects of MDR-reversing agents on the cytotoxicity of VCR
against MDR KB/V cell line
Compd
IC50 (nM)
Fold reversala
VCR
1230.48
218.02
7.30
––
VCR+compd 8 (5lM)
VCR+compd 8 (10lM)
VCR+compd 9 (5lM)
VCR+compd 9 (10lM)
VCR+compd 10 (5lM)
VCR+compd 10 (10lM)
VCR+taxinine (10lM)
VCR+verapamil (10lM)
5.64
168.56
63.32
439.46
3.78
19.43
2.80
1
the compounds 8, 9, 10 were confirmed by H NMR,
325.42
8.93
13C NMR, and FABMS.7
137.79
9.91
67.81
124.18
18.14
The taxinine analogues prepared in this study were
tested for their reversal activity in epidermoid KB hu-
man cancer cell line and its MDR subline KB/V cell.
Compound 9 was then further evaluated for their activ-
ity to increase accumulation of intracellular Rho.123
and in vivo sensitizing activity with human epidermoid
(KB, KB/V) cancer xenografts. Chemosensitivity in
vitro was measured by means of the 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
colorimetric assay performed in 96-well plates.8 The
concentration that caused 50% inhibition of cancer cell
growth against various cancer cell lines were expressed
as IC50 values. The results are presented in Table 1.
The reversal activity of target compounds were ex-
pressed reversal folds (IC50 value for vincristine (VCR)
of KB/V cell with the reversing agents divided by IC50
value for VCR of KB/V cell without the reversing
agents). Compounds 8–10 showed potent MDR reversal
activity in vitro in a dose-dependent way. In order to ob-
tain more meaningful relative activities, verapamil was
tested at the same time. Overexpression of P-gp was
the main cause of MDR in KB/V cell line.9 Further-
more, we measured the effect of compound 9 on P-gp
Cell survival was determined by MTT assay.
a The reversal activity of target compounds were expressed reversal
folds (IC50 value for VCR of KB/V cell with the reversing agents
divided by IC50 value for VCR of KB/V cell without the reversing
agents).
function by Rho.123 dye-efflux assay. Compound 9 re-
sulted in significantly increased intracellular levels of
Rho.123 in a dose-dependent way (Fig. 2).
The sensitizing activity of compound 9 was investigated
in nude mice. Experiments were carried out in the hu-
man tumor xenografts KB and MDR subline KB/V
(Fig. 3A and B). As shown in Figure 3A, VCR adminis-
tered at a dose of 0.5mg/kg ipresulted in significant tu-
mor growth inhibition (p< 0.01, day 24). On the other
hand, as shown in Figure 3B, VCR administered at a
dose of 0.5mg/kg ip potently produced any growth inhi-
bition in VCR-resistant KB/V tumor xenografts. The
same was true when orally treated with compound 9
(330mg/kg) alone. When treated concomitantly with

X. Zhao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4767–4770
4769
in multidrug-resistant KB/V cells and had stronger activ-
ity than that of verapamil. In addition, we found that
compound 9 given orally enhanced the chemotherapeutic
effect of VCR in KB/V-bearing mice. These taxinine ana-
logues (8–10) may be useful for the treatment of tumors
when they are used in combination with anticancer
agents, since they increase the sensitivity to anticancer
agents. These taxinine analogues (8–10) may interact di-
rectly with P-gpand inhibit the active efflux of antitumor
agents, thus overcome MDR in vitro and in vivo.
500
450
400
350
300
250
200
150
100
50
0
KB
KB/V
Vera. Compd.9 Compd.9 Compd.9
5µm 10µm 20µm
Acknowledgements
Figure 2. Accumulation of Rho.123 in KB and KB/V cells.
This work was supported by NNSFC (grant no.
30100230). The authors thank Ms. Liyan for her skillfull
technical assistance.
5000
4500
4000
contr.
VCR
3500
3000
2500
2000
1500
1000
500
References and notes
1. Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer
2002, 2, 48.
2. Thomas, H.; Coley, H. M. Cancer Control 2003, 10, 159.
3. Kobayashi, J.; Shigemori, H.; Hosoyama, H.; Chen, Z.;
Akiyama, S.; Naito, M.; Tsuruo, T. Jpn. J. Cancer Res.
2000, 91, 638.
0
0
10
20
30
Days Post Implant
(A)
4. Cheng, K. D. PCT. Int. Appl. WO 9406, 740 (CI C07035/
37), 1994.
4000
3500
3000
2500
2000
1500
1000
500
5. Ahn, Y. M.; Velde, D. G. V.; Georg, G. I. J. Org. Chem.
2002, 67, 7140.
contr.
VCR
6. Zhang, M.; Yin, D. L.; Guo, J. Y.; Liang, X. T. Chin.
Chem. Lett. 2002, 13, 501.
Compd.9
Compd.9+VCR
1
7. Compound 8: mp51–52 ꢁC, H NMR (500MHz, CDCl3, d
ppm): 7.35–7.24 (m, 5H, Ph), 6.08 (dd, 1H, J=12, 5.5Hz,
H-10), 5.48 (dd, 1H, J=2, 6.5Hz, H-2), 5.27 (s, 1H, H-20),
5.24 (t, 1H, J=2Hz, H-5), 4.83 (s, 1H, H-20), 3.55 (q, 2H,
J=17Hz, 20-CH2), 3.10 (d, 1H, J=6.5Hz, H-3), 2.84 (dd,
1H, J=7, 19.7Hz, H-14), 2.50 (dd, 1H, J=11.7, 15Hz, H-
9), 2.37 (d, 1H, J=19.5Hz, H-14), 2.24 (s, 3H, 10-OAc-
CH3), 2.62 (dd, 1H, J=2, 7Hz, H-1), 2.10 (s, 3H, 2-OAc-
CH3), 2.06 (s, 3H, 18-CH3), 1.95 (m, 1H, H-7), 1.80 (m, 3H,
2·6-H, H-9), 1.70 (s, 3H, 16-CH3), 1.29 (m, 1H, H-7), 1.15
(s, 3H, 17-CH3), 0.89 (s, 3H, 19-CH3); FABMS: 537.5; HR-
FABMS (Gly/m-NBA): found 537.2755, calcd 537.2852,
formula: C32H41O7.
0
0
10
20
30
(B)
Days Post Implant
Figure 3. (A) Inhibition effect of VCR on VCR-sensitive KB tumor in
athymic mice. There were six mice per group. VCR, 0.5mg/kg (m) was
administered ipas single agent on days 0, 4, 8, 12, 16, and 20. Control
animals (j) were not treated. (B) Inhibition effect of VCR combined
compound 9 on VCR-resistant KB/V tumor in athymic mice. VCR,
0.5mg/kg (m) ip, compound 9, 330mg/kg (j) orally was administered
as single agent on days 0, 4, 8, 12, 16, and 20. Combined therapy (s)
included VCR, 0.5mg/kg (m) ip, compound 9, 330mg/kg orally on
days 0, 4, 8, 12, 16, and 20. Control animals (r) were not treated.
Compound 9: mp117 ꢁC, 1H NMR (500M Hz, CDCl3, d
ppm): 7.33–7.21 (5H, Ph), 6.06 (dd, 1H, J=12.3, 6Hz, H-
10), 5.48 (dd, 1H, J=1.5, 6.7Hz, H-2), 5.28 (s, 1H, H-20),
5.24 (br s, 1H, H-5), 4.84 (s, 1H, H-20), 3.17 (d, 1H,
J=6Hz, H-3), 2.96 (t, 2H, J=8Hz, 20-CH2), 2.81 (dd, 1H,
J=6.5, 20Hz, H-14), 2.63 (m, 1H, 30-CH2), 2.53 (m, 1H, 30-
CH2), 2.50 (dd, 1H, J=12, 15Hz, H-9), 2.32 (d, 1H,
J=20Hz, H-14), 2.18 (s, 3H, 10-OAc-CH3), 2.16 (dd, 1H,
J=1.5, 6.5Hz, H-1), 2.12 (s, 3H, 2-OAc-CH3), 2.09 (s, 3H,
18-CH3), 1.91 (m, 1H, H-7), 1.78 (m, 3H, 2·H-6, H-9), 1.71
(s, 3H, 16-CH3), 1.28 (m, 1H, H-7), 1.15 (s, 3H, 17-CH3),
0.92 (s, 3H, 19-CH3); FABMS: 551.3 (M+1); HR-FABMS
(Gly/m-NBA): found 550.2946, calcd 550.2931, formula:
C33H42O7 +H+.
compound 9 (330mg/kg po), and VCR (0.5mg/kg ip) re-
sulted in significant growth inhibition (p <0.01, day 24).
The tumor growth inhibition observed with the combi-
nation in VCR-resistant KB/V tumor xenografts was
just like that of VCR in VCR-sensitive KB tumor xeno-
grafts (p > 0.05).
Compound 10: mp69–71 ꢁC, 1H NMR (500M Hz, CDCl3, d
ppm): 6.82–6.75 (m, 3H, Ph), 6.09 (dd, 1H, J=5.5, 12Hz,
H-10), 5.48 (dd, 1H, J=1.5, 6.5Hz, H-2), 5.27 (s, 1H,
H-20), 5.25 (br s, 1H, H-5), 4.82 (s, 1H, H-20), 3.77 (s, 3H,
50-OCH3), 3.76 (s, 3H, 20-OCH3), 3.59 (d, 1H, J=17Hz,
H-20), 3.41 (d, 1H, J=17Hz, H-20), 3.23 (d, 1H, J=6.5Hz,
In conclusion, three taxinine analogues (8–10) at 10lM
markedly reversed the resistance to VCR in KB/V cells
(overexpress P-gp), while taxinine (1) showed weak
effect. Compound 9 increased the Rho.123 accumulation

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X. Zhao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4767–4770
H-3), 2.81 (dd, 1H, J=7, 20Hz, H-14), 2.50 (dd, 1H,
0.91 (s, 3H, 19-CH3); FABMS: 596.3; HR-FABMS (Gly/m-
NBA), found 596.2970, calcd 596.2985, formula: C34H44O9.
8. Han, R. Research and Development of Anticancer Drugs and
Experimental Techniques; PUMC: Beijing, 1996; pp 284–286.
9. Zhang, X.; Zhang, F.; Ji, X.; Li, Z. Acta Pharm. Sinica
1994, 26, 246.
J=12.3, 14.7Hz, H-9), 2.39 (d, 1H, J=20Hz, H-14), 2.24 (s,
3H, 10-OAc-CH3), 2.16 (d, 1H, J=5.5Hz, H-1), 2.09 (s, 3H,
2-OAc-CH3), 2.07 (s, 3H, 18-CH3), 2.17–1.96 (m, 1H, H-7),
1.83 (m, 2H, 2·H-6), 1.77 (dd, 1H, J=14.7, 5.7Hz, H-9),
1.70 (s, 3H, 16-CH3), 1.28 (m, H-7), 1.14 (s, 3H, 17-CH3),