Synthesis and biological evaluation of new taxoids derived from 2-deacetoxytaxinine J

Article abstract of DOI:10.1016/j.bmcl.2006.12.101

A small library of 2-deacetoxytaxinine J (DAT-J) 1 derivatives was synthesised and tested in vitro for their reversal activity in human mammary carcinoma MDR cell line MCF7-R. One of the new taxoids showed to be active at 0.1 μM when tested in combination

Full text of DOI:10.1016/j.bmcl.2006.12.101

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1579–1583
Synthesis and biological evaluation of new taxoids
derived from 2-deacetoxytaxinine J
Maurizio Botta,a,* Silvia Armaroli,a,b Daniele Castagnolo,a
Gabriele Fontana,c Paula Perad and Ezio Bombardellic
a
`
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, via A. Moro, 53100 Siena, Italy
bPrassis, Istituto di ricerche Sigma-Tau, via Forlanini, 1 Settimo Milanese (MI), Italy
cIndena S.p.A., Milan, Italy
dRoswell Park Cancer Institute, Pharmacology and Therapeutics, Buffalo, USA
Received 21 November 2006; revised 19 December 2006; accepted 26 December 2006
Available online 8 January 2007
Dedicated to Professor Fulvio Gualtieri on the occasion of his 70th birthday
Abstract—A small library of 2-deacetoxytaxinine J (DAT-J) 1 derivatives was synthesised and tested in vitro for their reversal activ-
ity in human mammary carcinoma MDR cell line MCF7-R. One of the new taxoids showed to be active at 0.1 lM when tested in
combination with paclitaxel.
Ó 2007 Elsevier Ltd. All rights reserved.
Paclitaxel (TaxolÒ), a complex natural diterpene extract-
ed from Taxus brevifolia,1 and its derivative docetaxel
are two of the most important anticancer agents for
the treatment of ovarian and breast cancer.2 Clinical
use of these anticancer drugs has disclosed that paclit-
axel and docetaxel have a number of undesired side ef-
fects as well as multidrug resistance (MDR). MDR
induced by taxoid treatment in tumour cells is a signifi-
cant obstacle to the success of chemotherapy in many
cancers. In particular, MDR is a phenomenon whereby
tumour cells that have been exposed to one cytotoxic
agent develop cross resistance to a range of structurally
and functionally unrelated compounds. The drug resis-
tance developed in cancer cells may be mediated by a
number of mechanisms; in particular it has been related
to the overexpression in cancer cells of particular pro-
teins such as P-glycoprotein (P-gp) which extrude
hydrophobic anticancer compounds and maintain their
intracellular concentration below a cytotoxic level.3
Several natural and semisynthetic taxoids, devoid of
cytotoxicity and tubulin affinity, are powerful inhibitors
of P-gp activity, acting as efficient reversing agents and
allowing accumulation of paclitaxel in MDR cancer
cells.4 New taxanes exhibited >99% MDR reversal activ-
ity against breast cancer cell lines, at 1–3 lM level.3a
Among the natural taxoids, 2-deacetoxytaxinine J
(DAT-J) 1 emerged as the most active P-gp inhibitor
with a potency higher than that of verapamil;4a com-
pound 1 showed an increase of vincristine accumulation
(266%) in MDR 2780 AD cells as compared to verapa-
mil (254%). DAT-J 1 is extracted in low concentration
from several yew species, but it can be synthesised from
the natural alkaloid 20-deacetoxyaustropicatine (DAS) 2
available in multigram amounts from Taxus x media
Rehd. Cv. HicKsii.5 Moreover, unlike other cinnamates
related to taxine, 1 does not show cardiac toxicity,6 and
might thus serve as an important starting material for
the synthesis of new reversal agents. For this reason,
the development of a procedure, using 2-deacetoxytaxi-
nine J 1 as starting material for the preparation of new
bioactive taxinine analogues, would be significative
(see Figs. 1 and 2).
Herein the preparation of a small library of DAT-J 1
analogues is reported; these derivatives have been
subjected to biological screening as new potential
MDR-reversing agents against multidrug-resistant
breast tumour cell lines.
Keywords: Taxoids; 2-Deacetoxytaxinine J; Multidrug resistance
(MDR).
*
Corresponding author. Tel.: +39 0577 234306; fax: +39 0577
234333; e-mail: botta@unisi.it
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.101

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M. Botta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1579–1583
Table 1. Derivatives of DAT-J 1
OAc
AcO
OAc
Compound
R1
10
9
O
4a
TES
5
13
O
AcO
O
H
H
1
O
N
Boc
4b
4c
Figure 1. The structure of 2-deacetoxytaxinine J 1.
MeO
O
OMe
HO
Bz
R2
O
O
N
Boc
HO
O
MeO
HO HN
Boc
Norstatin Side
Chain
O
N
5
Boc
OMe
7a
MeO
Protected Norstatin
Side Chain
Figure 2. Free acids of protected norstatin side chain 5 and norstatin
side chain of ortataxel.
OMe
7b
7c
7d
7e
7f
Cinnamoyl
3-Nicotinoyl
Bz
2-Deacetoxytaxinine J (DAT-J) 1 was prepared in one
step from commercially available 2-deacetoxyaustropica-
tine (DAS) 2 by elimination with m-CPBA5 (Scheme 1).
DAT-J 1 was converted into 3 by removal of the cinna-
moyl group with NH2OH.5b,7 Compound 3, which pre-
sents a free hydroxyl group at C-5 position, was further
elaborated into a first series of derivatives 4a–c presenting
different side chains.4a,8 The choice of the side chains was
investigated on the basis of the literature data and
interesting and easily available scaffolds. Compound 4a
was previously synthesised and evaluated in vitro for its
cytotoxicity against human cancer cell lines (TFK-1) by
Horiguchi et al.9
Bn
Phenylcarbamoyl
O
7g
7h
OH
O
HO HN
Boc
O
Esterification of 3 with the protected norstatin side
chain of ortataxel 510,11 (5 equiv) and benzoylchloride
(6 equiv) affords, respectively, derivatives 4b and 4c in
high yields (see Scheme 1 and Table 1).
O
N
Boc
8a
MeO
OMe
OAc
H
AcO
OAc
AcO
OAc
OAc
8b
8c
Cinnamoyl
O
N
O
i
cinn
O
O
AcO
AcO
H
H
H
2 (DAS)
1 (DAT J)
HO HN
Boc
ii
OAc
OAc
AcO
AcO
OAc
OAc
iii
OR1
OH
AcO
AcO
Compound 3 was then converted into diol 6 by selective
hydrolysis with LiOH at 4 °C5b,7 (Scheme 2). Diol 6 pre-
sents two secondary hydroxyls at C-5 and at C-13 posi-
tions; on the basis of the steric hindrance at C-5
position, a series of derivatives 7a–h was synthesised in
high yield, introducing different side chains selectively
at C-13 position of 6 (Scheme 2, Table 1). This latter
compound was elaborated first into 7a and 7b,
H
H
H
H
4a-c
3
Scheme 1. Reagents and conditions: (i) 1.2 equiv m-CPBA, THF, rt;
(ii) 9.3 equiv NH2OHÆHCl, NaOAc, EtOH/H2O 1:1, reflux; (iii) 3 equiv
TESCl, imidazole, DMF (for 4a, R1 = TES, 77%); 5 equiv 5, EDC,
toluene, DMAP (for 4b, R1 = protected norstatin side chain, 75%); 6
equiv BzCl, Et3N, toluene (for 4c, R1 = Bz, 70%).

M. Botta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1579–1583
1581
OAc
H
OAc
compound 7e, which represents a further simplification
of the side chain of 7d, was synthesised treating starting
material 6 with benzyl bromide (2 equiv) in presence of
NaH. Reaction of 6 with phenylisocyanate (2 equiv) in
CH2Cl2 under reflux affords the carbamate 7f. Finally,
compound 7g was obtained in two steps, by esterifica-
tion of 6 with TBDMS-(R)-phenyllactic acid (2 equiv)
followed by removal of silyl group with TBAF. Com-
pound 7a, presenting at C-13 position the protected
norstatin side chain of ortataxel, was further elaborated
into 7h by hydrolysis with 0.1 N HCl.12
AcO
AcO
OAc
OAc
i
OH
OH
AcO
HO
H
H
H
3
6
ii
OAc
AcO
OAc
AcO
OAc
OAc
O
iii
BocHN
OH
R2O
OH
O
H
H
HO
H
H
7h
7a-g
Further elaboration of compounds 7a and 7b into deriv-
atives 8a and 8b was accomplished by esterification at
position C-5 with cinnamic acid. Hydrolysis of protected
norstatin side chain of 8a afforded also derivative 8c (see
Scheme 3 and Table 1).
Scheme 2. Reagents and conditions: (i) 7 equiv LiOHÆH2O, MeOH/
H2O 1:1, 4 °C; (ii) 2 equiv acid, EDC, toluene, DMAP (for 7a,
R2 = protected norstatin side chain, 85%; for 7b, R2 = cinnamoyl, 79%;
for 7c, R2 = 3-nicotinoyl, 75%); 2 equiv BzCl, Et3N, CH2Cl2 (for 7d,
R2 = Bz, 82%); 2 equiv BnBr, NaH, THF (for 7e, R2 = Bn, 88%); 2
equiv PhNCO, Et3N, CH2Cl2, reflux (for 7f, R2 = PhNCO, 91%); 2
equiv TBDMS-phenyllactic acid, EDC, toluene, DMAP and then
TBAF, THF (for 7g, R2 = phenyllactoyl, 65% for 2 steps); (iii) HCl
0.1 N, MeOH (91%).
The synthesis of new oxidised derivatives of 1 was then
examined; in particular five compounds derived from
epoxidation of DAT-J 1 and the taxinine 3 were
synthesised.5a Horiguchi et al. first reported the
epoxidation of 2-deacetoxytaxinine-J 1 and its deriva-
tives with m-CPBA, and evaluated the biological activity
of new compounds as potential cytotoxic agents against
human cancer cell lines (TFK-1).13 Kobayashi et al. also
reported the epoxidation of the 4(20)-exomethylene in
taxinine and taxinine A, and evaluated their activity as
MDR-reversing agents.14 We decided to oxidise 1 with
m-CPBA in the presence of NaOAc following the
reported procedures; the reaction afforded a mixture of
the three compounds 9a (12% yield), 9b (25% yield)
and 9c (52% yield) which were separated by chromatog-
raphy. Moreover epoxidation of taxane 3 under the
same conditions afforded only the two derivatives 10a
(28% yield) and 10b (57% yield) which were separated
by chromatography. The stereochemistry of 9a–c and
10a,b was unequivocally established by NOE experi-
ments and through a comparison with data reported in
the literature.13,14 (Scheme 4).
OAc
OAc
AcO
AcO
OAc
OAc
O
i
R2O
R2O
OH
O
H
H
H
H
7a-b
8a-c
Scheme 3. Reagents and conditions: (i) 5 equiv cinnamic acid, EDC,
toluene, DMAP (for 8a, R2 = protected norstatin side chain, 62%; for
8b, R2 = cinnamoyl, 60%;); 5 equiv cinnamic acid, EDC, toluene,
DMAP and then HCl 0.1 N, MeOH (for 8c, R2 = norstatin side chain,
54% for 2 steps).
respectively, by esterification with the protected norsta-
tin side-chain acid 5 and with cinnamic acid. Reaction of
6 with nicotinic acid (2 equiv) in presence of EDC and
DMAP affords 7c. Esterification of compound 6 with
benzoyl chloride (2 equiv) affords taxinine 7d while
OAc
H
AcO
O
OAc
O
O
AcO
H
OAc
AcO
O
+
9a
OAc
O
i
OAc
AcO
1 (DAT J)
OAc
+
O
O
O
AcO
H
H
O
AcO
H
9b
O
H
9c
OAc
AcO
O
OAc
OAc
O
AcO
OAc
i
3
+
OH
OH
AcO
AcO
H
H
O
H
H
10a
10b
Scheme 4. Reagents and condition: (i) 1.5 equiv m-CPBA, NaOAc, CH2Cl2, rt.

1582
M. Botta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1579–1583
Biological evaluation. The DAT-J 1 analogues synthe-
sised in this work were tested in vitro for their MDR
revertant activity in human mammary carcinoma cell
line MCF7-R, which was resistant to paclitaxel. The
compounds were tested at non-cytotoxic concentration
(i.e., 1.0 and 0.1 lM) in combination with paclitaxel.
The activity was expressed as IC50 (nM) that is the con-
centration that caused 50% inhibition of cancer cell
growth. The results are presented in Table 2.
for their reversal activity in human mammary carcinoma
MDR cell line MCF7-R. Among the new compounds,
7d showed to be very active at very low concentration;
7d presented a 67% paclitaxel IC50 reduction when test-
ed in combination with paclitaxel at 0.1 lM.
Acknowledgements
We gratefully acknowledge Professor Giovanni Appen-
dino for his helpful suggestions. We are indebted to
Indena S.p.A. (Milano, Italy) for finacial support.
Financial support from the Italian Ministero dell’Ist-
Introduction of different side chains or ester groups at
C-5 position results in loss of activity; in fact, com-
pounds 4b,c and 8a–c were found to be totally inactive.
The derivative 4a which presents a silyl group at C-5
showed potent activity in increasing the cytotoxicity of
paclitaxel against MCF7-R cell line at 1.0 lM. The
lowest concentration was inactive. At the opposite,
the introduction of a side chain or an ester function at
C-13 position seems to be crucial for the MDR activity;
in fact, compound 6 which presents free hydroxyl
groups at C-5 and C-13 positions showed low revertant
activity at the higher concentration tested (1.0 lM).
Compound 7h with deprotected norstatin side chain of
ortataxel at C-13 showed potent paclitaxel-MDR revert-
ant activity at 1.0 lM but became inactive at 0.1 lM.
Compounds 7b and 7g were active at both concentra-
tions tested; in particular 7d15 showed to be very active
at 0.1 lM. The epoxidised compounds 9a–c showed a
good activity at 1.0 lM concentration; among these,
the derivative 9a with an epoxide at C-11 and C-12
showed also a weak activity at lower concentration.
`
ruzione, dell’Universita
e
della Ricerca (PRIN
2005037820) is gratefully acknowledged. One of us
(M.B.) thanks the Merck Research Laboratories for
the 2002 Academic Development Programme (ADP)
Chemistry Award.
References and notes
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Farmaco 2002, 57, 385.
In conclusion, a small library of 2-deacetoxytaxinine J
(DAT-J) 1 derivatives was synthesised and tested in vitro
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mori, H.; Koiso, Y.; Iwasaki, S.; Sasaki, T.; Naito, M.;
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Table 2. Effects of MDR-reversing agents on the cytotoxicity of
paclitaxel against MCF7-R cell line
Compound Concentration Paclitaxel IC50 % paclitaxel
tested in combination (nM) MCF7-R IC50 reduction
with paclitaxel (lM)
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Barboni, L.; Torregiani, E.; Gabetta, B.; Zini, G.; Bom-
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39, 175.
4a
6
1.0
0.1
47 8.7
662 67
92
0
1.0
0.1
325 46
641 54
47
0
7b
7d
7g
7h
9a
9b
9c
1.0
0.1
45 4.0
386 78
92
35
1.0
0.1
31 8.0
204 52
95
67
1.0
0.1
66 8.7
463 59
89
24
7. Sun, D.-A.; Nikolakakis, A.; Sauriol, F.; Mamer, O.;
Zamir, L. Bioorg. Med. Chem. 2001, 9, 1985.
1.0
0.1
44 13
597 79
93
0
8. (a) Kobayashi, J.; Hosoyama, H.; Wang, X.; Shigemori, H.;
Sudo, Y.;Tsuruo, T. Bioorg. Med. Chem. Lett. 1998, 8, 1555;
(b) Hosoyama, H.; Shigemori, H.; Tomida, A.; Tsuruo, T.;
Kobayashi, J. Bioorg. Med. Chem. Lett. 1999, 9, 389.
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2000, 41, 3907.
10. Pratesi, G.; Laccabue, D. Drugs Future 2001, 26, 533.
11. Ortataxel is a taxane selected for preclinical development;
it shows a remarkably better bioavailability than paclitaxel
and docetaxel after oral administration and a wide activity
spectrum against various cancer types.
1.0
0.1
46 9.8
463 56
92
22
1.0
0.1
178 24
583 94
70
2
1.0
0.1
92 14
613 82
85
0
Paclitaxel None
593 41

M. Botta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1579–1583
1583
12. Appendino, G.; Bettoni, P.; Noncovich, A.; Sterner, O.;
Fontana, G.; Bombardelli, E.; Pera, P.; Bernacki, R. J.
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13. Horiguchi, T.; Nagura, M.; Cheng, Q.; Oritani, T.; Kudo,
T. Heterocycles 2000, 53, 2629.
(Na2SO4), filtered and evaporated to give the crude 7d.
The crude product was purified by flash chromatography
on silica gel, using AcOEt/petroleum ether 1:2 as eluant.
1
Yield: 82%. H NMR (CDCl3): d 8.08–8.02 (2H, m, Ph),
7.52–7.41 (3H, m, Ph), 6.31 (1H, d, J = 10.9 Hz, H-10),
5.85 (1H, d, J = 10.9 Hz, H-9), 5.87–5.85 (1H, m, H-13),
5.75 (1H, dd, J = 5.4, 11.5 Hz, H-7), 5.05 (1H, s, H-20),
4.75 (1H, s, H-20), 4.25 (1H, m, H-5), 3.30 (1H, m, H-3),
2.90 (1H, br s, OH), 3.03–2.84 (1H, m, H-14a), 2.36 (3H, s,
CH3-18), 2.05 (3H, s, CH3-10), 2.01 (3H, s, CH3-7), 1.96
(3H, s, CH3-9), 1.91–1.66 (5H, m, H-1, H-2a,b, H-6a,b),
1.59 (3H, s, CH3-17), 1.23–1.10 (1H, m, H-14b), 1.06 (3H,
s, CH3-16), 0.80 (3H, s, CH3-19). MS : 583 (M + 1), 605
(M + Na). Anal. Calcd for C33H42O9: C, 68.02; H, 7.27.
Found: C, 68.23; H, 7.42.
14. Hosoyama, H.; Shigemori, H.; In, Y.; Ishida, T.; Kobay-
ashi, J. Tetrahedron 1998, 54, 2521.
15. Experimental procedure for the synthesis of compound 7d.
Taxinine 6 (115 mg, 0.24 mmol) was dissolved in dry
CH2Cl2 (10 mL) and the resulting solution was cooled at
0 °C; Et3N (43 lL, 0.31 mmol), BzCl (34 lL, 0.29 mmol)
and a catalytic amount of DMAP were added and the
mixture was stirred at room temperature for 2 h. The
solution was diluted with CH2Cl2 and washed with
NaHCO3 and brine. The organic layers were dried