In vitro and in vivo anticancer activity of 2-deacetoxytaxinine J and synthesis of novel taxoids and their in vitro anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2009.04.022

The taxane diterpneoid 2-deacetoxytaxinine J (2-DAT-J) 1 has been isolated from the bark of Himalayan yew, Taxus baccata L. spp. wallichiana in a reasonably good yield (0.1%) and its anticancer activity against breast cancer cell lines (MCF-7 and MDA-MB-2

Full text of DOI:10.1016/j.ejmech.2009.04.022

European Journal of Medicinal Chemistry 44 (2009) 3947–3953
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
In vitro and in vivo anticancer activity of 2-deacetoxytaxinine J and synthesis
of novel taxoids and their in vitro anticancer activityq
K. Papi Reddy a, Hemant K. Bid b, V. Lakshma Nayak b, Preeti Chaudhary b, J.P. Chaturvedi a,
,
K.R. Arya c, Rituraj Konwar b, T. Narender a
*
a Medicinal and Process Chemistry Division, Central Drug Research Institute, Chattrar Manzil, Lucknow 226 001, Uttar Pradesh, India
b Endocrinology Division, Central Drug Research Institute, Chattrar Manzil, Lucknow 226 001, Uttar Pradesh, India
c Botany Division, Central Drug Research Institute, Chattrar Manzil, Lucknow 226 001, Uttar Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The taxane diterpneoid 2-deacetoxytaxinine J (2-DAT-J) 1 has been isolated from the bark of Himalayan
yew, Taxus baccata L. spp. wallichiana in a reasonably good yield (0.1%) and its anticancer activity against
breast cancer cell lines (MCF-7 and MDA-MB-231) and normal human kidney epithelial cell line (HEK-
293) has been studied. 2-DAT-J (1) showed significant in vitro activity against breast cancer cell line at
Received 8 October 2008
Received in revised form
12 February 2009
Accepted 14 April 2009
Available online 22 April 2009
a concentration of 20 mM and 10 mM in MCF-7 and MDA-MB-231 respectively. Few novel taxoids were
derived (7, 8 and 10–13) from the naturally occurring 2-DAT-J (1) and screened for their anticancer
activity. The structure–activity relationship studies indicated that the cinnamoyl group on C-5 and acetyl
group on C-10 are essential for the anticancer activity. 2-DAT-J (1) was also tested for its in vivo activity on
DMBA-induced mammary tumors in virgin female Sprague Dawley rats at a dose of 10 mg/kg body
weight orally for 30 days and showed significant regression in mammary tumors as compared to vehicle
treated group (p < 0.05).
Keywords:
2-Deacetoxytaxinine J (2-DAT-J)
Taxus wallichiana
Anticancer activity
Taxol
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
[4]. Ample approaches towards the semi-synthetic routes to taxol
have been developed.
Taxanes are an integral part of the treatment of multiple cancer
types, including breast and ovarian cancer. TaxolÒ (3) which
belongs to taxane class of compound, is a diterpenoid pseudo
alkaloid and was first isolated from the bark of the western yew,
Taxus brevifolia, by Wall and Wani’s group in collaboration with
National Cancer Institute (NCI) of USA, and is a particularly signif-
icant new lead for the treatment of cancer [1]. Thus far, it has been
approved by the FDA of US for the treatment of advanced ovarian
and metastatic breast cancer and is currently in phase II and III
clinical trials for lung and other cancers [2]. The outstanding
cytotoxic activity of taxol is believed to arise from its unique
propensity to hinder cell replication by preventing microtubules
from depolymerization [3]. The only source of taxol was from the
needles (0.033%) and bark (0.01) of the pacific yew tree T. brevifolia,
which cannot be considered as a renewable source because of its
slow growth and, therefore, the supply of taxol was quite limited
Apart from this, six total syntheses of taxol have been published
to date [5]. On the other hand, the supply of the key terpenoid
fragment baccatin III (2) (0.2%) and 10-deacetylbaccatin III (4)
(0.1%) is obtained from the leaves, a rapidly renewed resource of
Taxus baccata. The side chain attached to the main ring of baccatin
III is N-bezoyl-(2R,3S)-phenylisoserine (5) in the synthesis of taxol
(Fig. 1) [6].
The shortest route was reported by Wender et al., starting from
(1R)-(þ)-verbenone which involves 37 steps [5d] in an overall yield
of about 0.37%. The scarcity of taxol from the natural source and
economically not viable synthetic methods have led to search for
alternative compound isolable in useful quantities.
In continuation of our drug discovery program on anticancer
agents we isolated 2-DAT-J (1) in reasonably good yield (0.1%) from
the Indian T. baccata (spp. wallichiana). Chattopadhyay and co-
workers [7] isolated 1 from the same species. 2-DAT-J (1) was also
reported from several other species such as Taxus canadensis [8],
Taxus mairei [9], Taxus chinensis [10], T. brevifolia [11], Taxus cuspi-
date [12] and Taxus yunnanensis [13]. It exhibits cytotoxicity against
L1210 murine leukemia cells and KB human epidermoid carcinoma
cells and has effects on the Ca2þ induced microtubule depolymer-
ization [14]. Taxoid 1 devoid of cytotoxicity and tubulin affinity is
q
CDRI communication no. 7627.
* Corresponding author. Tel.: þ91 522 2612411; fax: þ91 522 2623405/2623938.
E-mail
addresses:
tnarender@rediffmail.com,
t_narendra@cdri.res.in
(T. Narender).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.022

3948
K.P. Reddy et al. / European Journal of Medicinal Chemistry 44 (2009) 3947–3953
AcO
O
OH
OAc
H
AcO
H
OAc
HO
O
AcO
O
H
O
AcO
O
HO
O
Ph
1
2
HO
O
OH
O
AcO
11
O
OH
7
Ph
NH
O
10
9
HO
12
13
6
5
8
Ph
3
O
4
O
H
HO
AcO
OH
14
2
O
O
1
H
HO
AcO
O
O
O
Ph
4
3
Ph
O
O
Ph
NH
O
N
Ph
OH
OH
6
5
Fig. 1. 2-Deacetoxytaxinine J (1); baccatin III (2); paclitaxel (3 TaxolÒ: BMS-181339); 10-deacetylbaccatin III (4); phenylisoserine (5 taxol C-13 side chain); Tamoxifen (6).
AcO
H
OAc
H
a powerful inhibitor of P-gp activity, acting as efficient reversing
agent in multi-drug resistant (MDR) cancer cells [15]. It increases
vincristine accumulation (266%) in MDR 2780 AD cells as compared
to verapamil (254%). Recently Botta and co-workers prepared 2-
DAT-J (1) in one step from commercially available 2-deacetox-
yaustropicatine (2-DAS) and synthesized a small library of 2-DAT-J
(1) analogues to develop potential MDR-reversing agents against
MDR-resistant tumor cell lines [16]. Several groups reported the in
vitro anticancer activity of 2-DAT-J (1), to our knowledge in vivo
anticancer activity in animal models has not yet been reported.
The present study was focused to develop alternative compound
for taxol. Towards this end, we here report the in vivo anticancer
activity of 2-DAT-J (1) for the first time and synthesis of few novel
taxoids and their in vitro anticancer activity.
AcO
H
OAc
H
OAc
OAc
(DHQD)2 PHAL, OSO4
O
O
OH
OH
AcO
AcO
NMO, tBuOH/H2O, rt.
O
O
8
1
Scheme 2. Dihydroxylation of 2-DAT-J (1).
Hydrolysis of 2-DAT-J (1) under basic conditions such as NaOH or
KOH led to mixture of several compounds. Therefore NH2OH$H2SO4
was used to regioselectively decinnamoylate 1 without disturbing
other acetyl groups [19]. Further we used this decinnamoylated
derivative 9 to substitute the cinnamoyl group with heterocyclic
acids to furnish 10–13 (Scheme 3).
2.2. Biology
2. Results and discussion
2-DAT-J (1), its derivatives 7, 8 and its analogues 9–15 were
screened for their in vitro anticancer activity against two breast
cancer cell lines, MCF-7 (ER-positive) and MDA-MB-231
(ER-positive) and a normal human kidney epithelial cell line
2.1. Chemistry
Chemical transformation of the active compound was carried
out to study the structure–activity relationship. Initially the selec-
tive hydrogenation of olefinic bond [17] of cinnamoyl group was
carried out on 2-DAT-J (1) using NaBH4, NiCl2$6H2O to obtain the
dihydro derivative 7 (Scheme 1).
To increase the hydrophilic character of the compound Sharp-
less asymmetric dihydroxylation [18] was carried out on 1 using
OsO4, which provided dihydroxylated derivative 8 (Scheme 2).
To find out the role of the cinnamoyl moiety in 2-DAT-J (1) we
attempted to remove the cinnamoyl group from 2-DAT-J (1).
AcO
H
OAc
H
AcO
H
OAc
NH2OH.H2SO4
OAc
OAc
O
AcO
H2O:THF:EtOH
(1:1:1)
reflux,6 h
AcO
H
O
OH
9
1
Appropriateacid
DCC-DMAP
CH2Cl2, rt., 24 h.
AcO
OAc
OAc
O
AcO
H
AcO OAc
AcO OAc
OAc
NaBH4/ NiCl2. 6H2O
MeOH, rt, 30 min.
O
R
OAc
O
H
N
O
O
AcO
AcO
R=
O
H
H
H
7
N
N
N
H
11
12
13
1
10
Scheme 1. Regioselective hydrogenation of 2-DAT-J (1) by using NaBH4/NiCl2$6H2O.
Scheme 3. Synthesis of ester analogues of 2-DAT-J (1).

K.P. Reddy et al. / European Journal of Medicinal Chemistry 44 (2009) 3947–3953
3949
Table 1
Anticancer activity of 2-deacetoxytaxinine J (2-DAT-J) 1 and its derivatives.
Entry
Compound
IC50
MCF-7
(m
M)c
MDA-MB-231
HEK-293
AcO
OAc
OAc
O
AcO
1
20
10
>50
O
H
H
1
AcO
OAc
OAc
O
AcO
2
15
20
10
O
H
H
7
AcO
OAc
OAc
O
OH
AcO
3a
>50
13
>50
O
H
OH
H
8
AcO
OAc
OAc
AcO
4
>50
>50
>50
OH
H
H
9
AcO
H
OAc
OAc
O
AcO
5
34
>50
>50
O
H
10
N
AcO
OAc
OAc
O
AcO
6
28
>50
>50
O
N
H
H
11
AcO
OAc
OAc
O
AcO
7
39
>50
>50
O
H
H
12
N
(continued on next page)

3950
K.P. Reddy et al. / European Journal of Medicinal Chemistry 44 (2009) 3947–3953
Table 1 (continued )
Entry
Compound
IC50 (m
M)c
MCF-7
MDA-MB-231
HEK-293
AcO
H
OAc
H
OAc
O
AcO
8
>50
>50
>50
O
13
N
b
9
10
Tamoxifen (6)
Taxol (3)
12
24
b
b
43
a
Stereochemistry was not determined.
Not done.
Values are mean of three independent experiments.
b
c
(HEK-293) and the results are presented in Table 1 [20,21]. The
3. Conclusion
natural compound 2-DAT-J (1) showed significant activity against
breast cancer cell line at a concentration of 20
MCF-7 and MDA-MB-231 respectively. The marketed drugs such as
Tamoxifen showed an IC50 of 12 M (MCF-7) and 24 M (MDA-MB-
231). The dihydro derivative 7 of 2-DAT-J is more potent than the
parent compound (1) and also other analogues against MCF-7 cell
line, however, 7 showed significant cytotoxicity towards normal
m
M and 10
m
M in
In conclusion, we have isolated a taxane diterpenoid 2-DAT-J (1)
from the bark of T. baccata (spp. wallichiana) in reasonably good
yield and prepared few novel taxoid derivatives and studied their
anticancer activity. The natural compound 2-DAT-J (1) has
comparable in vitro activity with marketed drug, Tamoxifen. 2-DAT-
J (1) also induced significant tumor regression in DMBA-induced
mammary tumors in Sprague Dawley rats at a dose of 10 mg/kg
body weight by oral route. The structure–activity relationship
indicated that the cinnamoyl group on C-5 is essential for the
anticancer activity (entries 1 and 4 in Table 1). Replacing the cin-
namoyl group with other heterocyclic acids as in 10–13 did not
improve the activity.
m
m
cell line (HEK-293). The compound 7 has an IC50 of 15
and 10 M against MCF-7, MDA-MB-231 and HEK-293 respectively.
The more hydrophilic dihydroxylated compound exhibited
mM, 20 mM
m
8
significant activity against only MDA-MB-231 cell line. Removal of
cinnamoyl group from 1 to provide 9 completely diminished the
activity in all the cell lines tested, which indicates the importance of
cinnamoyl group in the biological activity. Only three analogues, in
which the cinnamoyl group was replaced with heterocyclic
4. Experimental section
aromatic acids such as 10 (IC50 of 34
mM), 11 (IC50 of 28 mM), 12 (IC50
of 39 M) exhibited moderate activity against MCF-7 cell lines.
Nicotinic acid derivative 13 did not show significant activity in all
the tested cell lines.
m
4.1. General methods
Melting points were recorded on Buchi-530 capillary melting
point apparatus and are uncorrected. IR spectra were recorded on
Perkin–Elmer AC-1 spectrometer. 1H NMR spectra were run on
Bruker Advance DPX 300 MHz and 200 MHz in CDCl3. 13C NMR
spectra were recorded at 75 MHz and 50 MHz in CDCl3. Chemical
shifts are reported as values in ppm relative to CHCl3 (7.26) in CDCl3
and TMS was used as internal standard. ESI mass spectra were
recorded on JEOL SX 102/DA-6000. Chromatography was executed
with silica gel (60–120 mesh) using mixtures of ethyl acetate and
hexane as eluants. Ethyl acetate and hexane were dried and puri-
fied by distillation prior to use.
2-DAT-J (1) was also tested for its in vivo activity on DMBA-
induced mammary tumors in virgin female Sprague Dawley rats at
a dose of 10 mg/kg body weight orally for 30 days and showed
significant regression in mammary tumors as compared to vehicle
treated group (p < 0.05) (Fig. 2) [22].
4.2. Isolation of 2-deacetoxytaxinine J (1)
The plant material (10 kg of stem bark) of T. baccata (spp. wall-
ichiana) belongs to the family of Taxaceae. It was collected in the
month of June, 2002 from Dhakudi, Bhageshwar district, Uttar-
anchal state, India by the Botany Division of CDRI, Lucknow and
a voucher specimen (No. KRA 23932) was deposited in the
departmental herbarium of CDRI. The bark of the plant was
extracted with 4 L of ethyl alcohol four times in a percolator. The
resultant alcoholic extract was (16 L) combined and concentrated
under reduced pressure to give alcoholic extract. This was frac-
tionated with chloroform and butanol successively. The resultant
chloroform fraction was subjected to conventional silica gel column
chromatography using hexane and ethyl acetate solvent system to
Fig. 2. Effect of 2-DAT-J (1) on mammary tumors of female Sprague Dawley rats.

K.P. Reddy et al. / European Journal of Medicinal Chemistry 44 (2009) 3947–3953
3951
give 2-deacetoxytaxinine J (1). Compound 1 was characterized by
using 1H NMR, 13C NMR, IR and mass spectral data and comparing
with literature data.
170.3, 169.7, 146.1, 140.2, 137.5, 135.2, 129.0, 128.8 (2C), 127.0 (2C),
117.4, 76.8, 76.4, 75.7, 74.6, 71.9, 70.9, 70.5, 46.6, 40.7, 39.8, 37.9,
34.4, 31.8, 31.5, 27.9, 27.6, 21.8, 21.4, 21.2, 21.1, 15.8, 13.7; MS (ESI)
m/z 708.4 (M þ 23)þ.
Mp: 146–147 ꢀC; IR (KBr) 2991, 2947, 2878, 1742, 1632, 1438,
1375, 1243, 1158, 1021, 770 cmꢁ1; 1H NMR (CDCl3, 300 MHz)
d 7.77
(d, J ¼ 16.1 Hz, 1H), 7.49 (m, 2H), 7.40 (m, 3H), 6.57 (d, J ¼ 16.1 Hz,
1H), 6.30 (d, J ¼ 11.1 Hz, 1H), 5.94 (d, J ¼ 11.1 Hz, 1H), 5.81
(t, J ¼ 8.4 Hz, 1H), 5.69 (dd, J ¼ 11.3, 5.8 Hz, 1H), 5.57 (s, 1H), 5.38
(s, 1H), 5.02 (s, 1H), 3.30 (d, J ¼ 4.2 Hz, 1H), 2.72 (m, 1H), 2.33 (s, 3H),
2.07 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.94–1.79 (m, 6H), 1.71 (s, 3H),
1.63 (s, 3H), 1.10 (s, 3H), 0.88 (s, 3H); 13C NMR (CDCl3, 75 MHz)
4.5. Acetic acid 7,10,13-triacetoxy-5-hydroxy-8,12,15,15-
tetramethyl-4-methylene-tricyclo[9.3.1.03,8]pentadec-11-en-9-yl
ester (9)
To a magnetically stirred solution of 2-deacetoxytaxinine J (1)
(500 mg, 0.7 mmol) in a mixture of solvents (H2O:THF:EtOH
(1:1:1), 10 mL) was added hydroxylamine$sulphuricacid salt
(250 mg, 3,5 mmol) and triethylamine (4.9 mmol) at rt. The whole
solution was stirred for 6 h under reflux. Solvents were removed by
vacuum, then the reaction mixture was dissolved in water
(100 mL), extracted with ethyl acetate (3 ꢂ100 mL). The combined
organic layer was dried over anhyd Na2SO4 and evaporated under
reduced pressure. Then the crude product was chromatographed
on silica gel to afford the desired compound 9 (363 mg, 89%); Mp:
188–190 ꢀC; IR (KBr) 3548, 2984, 2942, 1743, 1654, 1439, 1374, 1249,
d
170.9, 170.5, 170.1, 169.5, 166.4, 146.6, 146.0, 137.5, 135.3, 134.4,
130.8, 129.2 (2C), 128.3 (2C), 118.7, 116.2, 77.0, 75.1, 71.9, 70.8, 70.3,
46.6, 40.4, 39.6, 37.7, 34.8, 32.1, 31.4, 27.5, 21.7 (2C), 21.2 (2C), 21.1,
15.5, 13.4; MS (ESI) m/z 673.1 (M þ 23)þ.
4.3. 3-Phenyl-propionic acid 7,9,10,13-tetraacetoxy-8,12,15,15-
tetramethyl-4-methylene-tricyclo[9.3.1.03,8]pentadec-11-en-5-yl
ester (7)
To a magnetically stirred solution of 2-deacetoxytaxinine J (1)
(250 mg, 0.38 mmol) in methanol (25 mL) was added gradually
NiCl2$6H2O (46 mg, 0.19 mmol) at rt. When the clear solution
acquired a greenish color, the whole reaction mixture was brought
to 5 ꢀC and NaBH4 (21 mg, 0.57 mmol) was added portionwise.
After addition of NaBH4, the whole solution was stirred for 30 min
at 5 ꢀC to rt. Methanol was removed by vacuum, and then the
reaction mixture was dissolved in ethyl acetate and neutralized
with 10% HCl solution, the organic layer was washed with water,
dried over anhyd Na2SO4 and evaporated under reduced pressure.
Then the crude product was chromatographed on silica gel to afford
the desired compound 7 (168 mg, 67%): IR (KBr) 2929, 1737, 1596,
1136, 1024, 758 cmꢁ1 1H NMR (CDCl3, 300 MHz)
; d 6.25
(d, J ¼ 11.0 Hz,1H), 5.81 (d, J ¼ 11.0 Hz,1H), 5.70 (m, 2H), 5.17 (s, 1H),
5.83 (s, 1H), 4.29 (s, 1H), 3.20 (d, J ¼ 4.8 Hz, 1H), 2.76 (m, 1H), 2.19
(s, 3H), 2.05 (s, 6H), 2.07 (s, 3H), 1.97 (s, 3H), 1.99–1.67 (m, 6H), 1.56
(s, 3H), 0.98 (s, 3H), 0.80 (s, 3H); 13C NMR (CDCl3, 75 MHz)
d 170.5,
170.2, 169.8, 169.4, 151.6, 137.9, 136.1, 112.7, 73.5, 72.3, 70.2, 69.9,
46.9, 39.8, 39.1, 36.2, 35.6, 32.5, 32.3, 29.8, 27.1, 26.4, 21.6, 21.2, 21.1,
21.0, 16.1, 12.7; MS (ESI) m/z 543.0 (M þ 23)þ.
4.6. 3-Pyridin-3-yl-acrylic acid 7,9,10,13-tetraacetoxy-8,12,15,15-
tetramethyl-4-methylene-tricyclo[9.3.1.03,8]-pentadec-11-en-5-yl
ester1(10)
1438, 1243, 1163, 1022, 767 cmꢁ1; 1H NMR (CDCl3, 300 MHz)
d 7.29
(m, 2H), 7.20 (m, 3H), 6.26 (d, J ¼ 11.0 Hz, 1H) 5.91 (d, J ¼ 11.0 Hz,
2H), 5.55 (dd, J ¼ 11.3, 5.3 Hz, 1H), 5.42 (t, J ¼ 3.3 Hz, 1H), 5.29
(s, 1H), 4.96 (s, 1H), 2.99 (t, J ¼ 7.8 Hz, 2H), 2.89 (d, J ¼ 5.6 Hz, 1H),
2.85–2.56 (m, 3H), 2.18 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H),
1.93–1.72 (m, 6H), 1.67 (s, 3H), 1.64 (s, 3H), 1.14 (s, 3H), 0.86 (s, 3H);
To a magnetically stirred solution of compound 9 (250 mg,
0.5 mmol), DCC (5 mmol) and DMAP (5 mmol) in dry CH2Cl2
(10 mL) was added gradually (E)-3-(pyridin-3-yl)acrylic acid
(1 mmol) at rt. The whole solution was stirred for 24 h at rt. The
reaction mixture was cooled, filtered and washed with dry and cold
CH2Cl2, the filtrate was evaporated under reduced pressure. Then
the crude product was chromatographed on silica gel to afford the
desired compound 10 (72%): IR (KBr) 3021, 2928, 2855, 1743, 1654,
13C NMR (CDCl3, 50 MHz)
d 172.4, 171.0, 170.7, 170.2, 169.7, 147.1,
140.5, 137.8, 134.9, 129.1 (2C), 128.7 (2C), 126.8, 116.1, 77.2, 75.2,
72.0, 70.9, 70.5, 46.5, 39.8, 40.8, 38.1, 36.9, 34.4, 31.7, 31.5, 31.4,
27.9, 27.8, 21.8, 21.4, 21.3, 21.3, 15.5, 13.7; MS (FAB) m/z 593
(M ꢁ OAc (59))þ.
1523, 1424, 1372, 1217, 1046, 757 cmꢁ1 1H NMR (CDCl3, 300 MHz)
;
d
8.76 (d, J ¼ 1.1 Hz, 1H), 8.64 (dd, J ¼ 4.6, 1.3 Hz, 1H), 7.83 (m, 1H),
7.76 (d, J ¼ 16.1 Hz, 1H), 7.37 (m, 1H), 6.65 (d, J ¼ 16.1 Hz, 1H), 6.29
(d, J ¼ 11.3 Hz, 1H), 5.95 (d, J ¼ 11.3 Hz, 1H), 5.81 (t, J ¼ 8.7 Hz, 1H),
5.66 (dd, J ¼ 10.9, 5.2 Hz, 1H), 5.59 (s, 1H), 5.40 (s, 1H), 5.04 (s, 1H),
3.03 (d, J ¼ 4.5 Hz, 1H), 2.76 (m, 1H), 2.32 (s, 3H), 2.08 (s, 3H), 2.04
(s, 3H), 1.99 (s, 3H), 1.95–1.79 (m, 6H), 1.73 (s, 3H), 1.64 (s, 3H), 1.10
4.4. 2,3-Dihydroxy-3-phenyl-propionic acid 7,9,10,13-tetraacetoxy-
8,12,15,15-tetramethyl-4-methylene-tricyclo[9.3.1.03,8]pentadec-
11-en-5-yl ester (8)
To a magnetically stirred solution of 2-deacetoxytaxinine J (1)
(250 mg, 0.38 mmol) and (DHDQ)2 PHAL (5 mol%) in tBuOH/H2O
mixture (1:1, 10 mL) were added gradually NMO (50 wt% in water,
1.1 mmol) and OsO4 (9 mg, 0.038 mmol). The pH was adjusted to 5
by addition of 2 N H2SO4, and the reaction mixture was stirred for
48 h at room temperature. Sodium sulfite (100 mg) was added, and
the reaction mixture was stirred for an additional hour. It was then
extracted with ethyl acetate (3 ꢂ 25 mL), the organic layer was
washed with water, dried over anhyd Na2SO4 and evaporated under
reduced pressure. Then the crude product was chromatographed
on silica gel to afford the desired compound 8 (110 mg, 42%). IR
(s, 3H), 0.89 (s, 3H); 13C NMR (CDCl3, 75 MHz)
d 171.0, 170.7, 170.3,
169.7, 165.9, 151.8, 151.8, 150.2, 146.5, 142.4, 137.4, 135.6, 134.6,
130.3, 124.2, 120.9, 116.6, 77.4, 75.6, 71.2, 70.4, 46.7, 40.5, 37.9, 34.9,
32.3, 31.6, 31.4, 27.6, 26.6, 25.8, 21.9, 21.4, 21.3, 15.7, 13.6; MS (ESI)
m/z 652.2 (M þ H)þ, 674.2 (M þ 23)þ.
4.6.1. Pyridin-2-yl-acetic acid 7,9,10,13-tetraacetoxy-8,12,15,15-
tetramethyl-4-methylene-tricyclo[9.3.1.03,8]pentadec-11-en-5-yl
ester (11)
Mp: 84–86 ꢀC; IR (KBr) 3329, 2931, 2853, 1741, 1628, 1576, 1438,
1374, 1243, 1157, 1023, 754 cmꢁ1; 1H NMR (CDCl3, 300 MHz)
d 8.56
(KBr) 2943, 2858, 1739, 1654, 1375, 1238, 1115, 1025, 770 cmꢁ1
NMR (CDCl3, 200 MHz)
;
1H
(d, J ¼ 4.3 Hz, 1H), 7.69 (dt, J ¼ 7.7, 1.7 Hz, 1H), 7.32 (d, J ¼ 7.7 Hz, 1H),
7.21 (m, 1H), 6.27 (d, J ¼ 11.1 Hz, 1H), 5.97 (d, J ¼ 8.6 Hz, 1H), 5.93
(d, J ¼ 11.1 Hz, 1H), 5.99 (dd, J ¼ 11.5, 5.14 Hz, 1H), 5.48 (s, 1H), 5.31
(s, 1H), 4.99 (s, 1H), 4.00 (s, 1H), 2.98 (d, J ¼ 5.4 Hz,1H), 2.68 (m, 1H),
2.19 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.96–
1.68 (m, 7H), 1.65 (s, 3H), 1.15 (s, 3H), 0.87 (s, 3H); 13C NMR (CDCl3,
d
7.37 (m, 5H), 6.25 (d, J ¼ 11.0 Hz, 1H), 5.90
(d, J ¼ 11.0 Hz, 2H), 5.46 (d, J ¼ 11.0 Hz, 2H), 5.03 (s, 2H), 4.53 (s, 1H),
4.30 (m, 1H), 2.80 (s, 1H), 2.58 (m, 1H), 2.21 (s, 3H), 2.08 (s, 3H), 2.03
(s, 3H), 1.98 (s, 3H), 1.94–1.79 (m, 6H), 1.76 (s, 3H), 1.62 (s, 3H), 1.11
(s, 3H), 0.91 (s, 3H); 13C NMR (CDCl3, 50 MHz)
d 172.5, 171.3, 170.6,

3952
K.P. Reddy et al. / European Journal of Medicinal Chemistry 44 (2009) 3947–3953
75 MHz)
d
170.8, 170.4, 170.2, 169.9, 169.4, 154.2, 149.8, 146.8, 137.7,
instillation of 10 mg/100 g body weight, of 7,12-dimethylbenzan-
thracene (DMBA) under prescribed anesthesia. Animals were
palpated biweekly for tumor development, tumor size measured
and were grouped into Group A (n ¼ 3): orally with 10 mg/kg body
weight of 2-DAT-J (1) in 0.5 mL of vehicle daily for 30 days; Group B
(n ¼ 3): orally with 10 mg/kg body weight of Tamoxifen citrate in
0.5 mL of vehicle daily for 30 days; and Group C (n ¼ 3): orally
0.5 mL vehicle only for 30 days. Tumor sizes were measured with
Vernier calipers on two days interval.
137.0, 134.7, 123.9, 122.5, 116.1, 76.8, 75.5, 71.9, 70.9, 70.3, 46.4, 43.9,
40.7, 39.7, 37.9, 34.2, 31.6, 31.4, 27.8, 27.7, 21.7, 21.6, 21.2, 21.0, 15.2,
13.6; MS (ESI) m/z 640.1 (M þ H)þ.
4.6.2. 3-Pyridin-3-yl-propionic acid 7,9,10,13-tetraacetoxy-
8,12,15,15-tetramethyl-4-methylene-tricyclo[9.3.1.03,8]pentadec-
11-en-5-yl ester (12)
Mp: 90–92 ꢀC; IR (KBr) 3021, 2929, 2853, 1734, 1623, 1579, 1432,
1373, 1217, 1024, 757 cmꢁ1 1H NMR (CDCl3, 300 MHz)
; d 8.49
(d, J ¼ 3.5 Hz, 2H), 7.56 (d, J ¼ 7.8, Hz, 1H), 7.25 (m, 1H), 6.24
(d, J ¼ 11.2 Hz, 1H), 5.92 (m, 2H), 5.22 (dd, J ¼ 11.3, 5.3 Hz, 1H), 5.42
(t, J ¼ 3.2 Hz, 1H), 5.29 (s, 1H), 4.97 (s, 1H), 3.00 (m, 3H), 2.88
(d, J ¼ 5.3 Hz,1H), 2.78 (m, 2H), 2.17 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H),
1.99 (s, 3H), 1.93–1.80 (m, 6H), 1.77 (s, 3H), 1.64 (s, 3H), 1.14 (s, 3H),
Acknowledgements
Authors are grateful to the Director, CDRI, Lucknow, India for
constant encouragement for the drug developing program on
anticancer agents, Rajesh (BIT, Ranchi) for technical support and
SAIF for spectral data.
0.88 (s, 3H); 13C NMR (CDCl3, 75 MHz)
d 171.6, 170.5, 170.4, 170.0,
169.4, 150.0, 148.2, 146.7, 137.4, 136.0, 135.7, 134.9, 123.9, 116.0, 76.9,
75.3, 72.7, 70.9, 70.2, 46.3, 40.7, 39.7, 38.0, 36.8, 34.3, 32.9, 31.5, 31.3,
27.7, 27.6, 21.6, 21.3, 21.2, 21.0, 15.2, 13.5; MS (ESI) m/z 654.2
(M þ H)þ, 676.2 (M þ 23)þ.
Appendix. Supplementary data
Spectra (1H NMR, 13C NMR and Mass) of all the compounds
associated with this article are available as supplementary data.
Supplementary data associated with this article can be found in the
online version, at doi:10.1016/j.ejmech.2009.04.022.
4.6.3. Nicotinic acid 7,9,10,13-tetraacetoxy-8,12,15,15-tetramethyl-
4-methylene-tricyclo[9.3.1.03,8]pentadec-11-en-5-yl ester (13)
Mp: 166–168 ꢀC; IR (KBr) 2952,1743,1627,1590,1437,1375,1238,
1115,1025, 745 cmꢁ1; 1H NMR (CDCl3, 300 MHz)
d
9.34 (d, J ¼ 1.7 Hz,
1H), 8.81 (dd, J ¼ 4.8, 1.5 Hz, 1H), 8.38 (dt, J ¼ 7.9, 1.9 Hz, 1H), 7.49
(dd, J ¼ 7.9, 4.9 Hz, 1H), 6.65 (d, J ¼ 16.1 Hz, 1H), 6.27(d, J ¼ 11.3 Hz,
1H), 5.96 (d, J ¼ 11.3 Hz,1H), 5.76 (m,1H), 5.65 (t, J ¼ 2.8 Hz,1H), 5.55
(s, 1H), 5.18 (s, 1H), 2.88 (d, J ¼ 4.7 Hz, 1H), 2.64 (m, 1H), 2.20 (s, 3H),
2.11 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H), 1.95–1.78 (m, 3H),
1.63 (s, 3H), 1.09 (s, 3H), 1.08 (s, 3H), 0.91 (s, 3H); 13C NMR (CDCl3,
References
[1] (a) J. Crown, M. O’Leary, Lancet 335 (2000) 1176–1178;
(b) M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon, A.T. McPhail, J. Am. Chem. Soc.
93 (1971) 2325–2327.
[2] (a) E.K. Rowinsky, L.A. Cazenave, R.C. Donehower, J. Natl. Cancer Inst. 82 (1990)
1247–1259;
75 MHz)
d 170.6, 170.5, 170.2, 169.5, 165.1, 153.9, 151.4, 146.0, 137.7,
137.4, 135.1, 126.6, 123.8, 118.1, 77.0, 76.9, 71.8, 70.5, 70.2, 46.5, 40.4,
39.6, 38.2, 34.7, 32.0, 31.3, 27.6, 27.5, 21.7, 21.7, 21.0, 19.9, 15.4, 13.8;
MS (ESI) m/z 626.2 (M þ H)þ, 648.1 (M þ 23)þ.
(b) W.J. Slichenmyer, D.D. Von Hoff, J. Clin. Pharmacol. 30 (1990) 770–788;
(c) W.J. Slichenmyer, D.D. Von Hoff, Anticancer Drugs 2 (1991) 519–530;
(d) M. Suffness, Ann. Rep. Med. Chem. 28 (1993) 305.
[3] (a) P.B. Schiff, J. Fant, S.B. Horwitz, Nature 277 (1979) 665–667;
(b) S.B. Horwitz, Trends Pharmacol. Sci. 13 (1992) 134–136;
(c) J.M. Andreu, J.F. Diaz, J. Garcia de Ancos, R. Gil, F.J. Mendrano, E. Nogales,
E. Pantos, E. Towns-Andrews, J. Mol. Biol. 226 (1992) 169–184;
(d) J.F. Diaz, J.M. Andreu, Biochemistry 32 (1993) 2747–2755;
(e) M.A. Jordan, R.J. Toso, D. Thrower, L. Wilson, Proc. Natl. Acad. Sci. U.S.A. 90
(1993) 9552–9556;
4.7. Cell lines
MCF-7 is an estrogen receptor positive breast cancer cell line
derived from pleural effusion and most commonly used cell line for
screening of anticancer breast agents [20], whereas MDA-MB-231 is
an estrogen receptor negative model of aggressive breast cancer.
HEK-293 is a transformed normal epithelial cell line originally
derived from human fetal kidney epithelium.
(f) I. Arnal, R.H. Wade, Curr. Biol. 5 (1995) 900–908;
(g) E. ter Har, R.J. Kowalski, E. Hamel, C.M. Lin, R.E. Longley, S.P. Gunasekara,
H.S. Rosenkranz, B.W. Day, Biochemistry 35 (1996) 243–250;
(h) D.T. Hung, J. Chen, S.L. Schreiber, Chem. Biol. 3 (1996) 287–293.
[4] N.C. Wheeler, K. Jech, S. Masters, S.W. Brobst, A.B. Alvarado, A.J. Hoover,
K.M. Snader, J. Nat. Prod. 55 (1992) 432–440.
[5] (a)R.A.Holton,H.B. Kim,C.Somoza,F. Liang, R.J.Biediger, P.D. Boatman, M.Shindo,
C.C. Smith, S. Kim, H. Nadizadeh, Y. Suzuki, C. Tao, P. Vu, S. Tang, P. Zhang,
K.K. Murthi, L.N. Gentile, J.H. Liu, J. Am. Chem. Soc. 116 (1994) 1599–1600;
(b) K.C. Nicolaou, H. Ueno, J.J. Liu, P.G. Nantermet, Z. Yang, J. Renaud,
K. Paulvannan, R. Chadha, J. Am. Chem. Soc. 117 (1995) 653–656;
(c) S.J. Danishefsky, J.J. Masters, W.B. Young, J.T. Link, L.B. Snyder, T.V. Magee,
D.K. Jung, R.C.A. Isaacs, W.G. Bornmann, C.A. Alaimo, C.A. Coburn, M.J. Di Grandi, J.
Am. Chem. Soc. 118 (1996) 2843–2859;
4.8. In vitro assay for anticancer activity
The cytotoxic activity of the compounds was determined using
MTT assay [16]. 1 ꢂ104 cells/well were seeded in 200
ml DMEM,
supplemented with 10% FBS in each well of 96-well microculture
plates and incubated for 24 h at 37 ꢀC in a CO2 incubator.
Compounds, diluted to the desired concentrations in culture
medium, were added to the wells with respective vehicle control.
(d) P.A. Wender, N.F. Badham, S.P. Gonway, P.E. Floreancing, T.E. Glass, J.B. Houze,
N.E. Krauss, D. Lee, D.G. Marquess, P.L. McGrane, W. Meng, M.G. Natchus,
A.J. Shuker, J.C. Sutton, R.E. Taylor, J. Am. Chem. Soc. 119 (1997) 2757–2758;
(e) T. Mukaiyama, I. Shiina, H. Iwadare, M. Saitoh, T. Nishimura, N. Ohkawa,
H. Sakoh, K. Nishimura, Y. Tani, M. Hasegawa, K. Yamada, K. Saitoh, Chem.dEur. J.
5 (1999) 121–161;
After 18 h of incubation, 10 ml MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to each
well and the plates were further incubated for 4 h. Then the
supernatant from each well was carefully removed, formazon
(f) H. Kusama, R. Hara, S. Kawahara, T. Nishimori, H. Kashima, N. Nakamura,
K. Morihara, I. Kuwajima, J. Am. Chem. Soc. 122 (2000) 3811–3820.
[6] (a) J.N. Denis, A.E. Greene, D. Guenard, F. Gueritte-Voegelein, L. Mangatal,
P. Potier, J. Am. Chem. Soc. 110 (1988) 5917–5919;
crystals were dissolved in 100 ml of DMSO and absorbance at
(b) L. Mangatal, M.T. Adeline, A.E. Greene, D. Guenard, F. Gueritte-Voegelein,
P. Potier, Tetrahedron 45 (1989) 4177–4190;
540 nm wavelength was recorded.
(c) H.-J. Ha, G.-S. Park, Y.-G. Ahn, G.S. Lee, Bioorg. Med. Chem. Lett. 8 (1998)
1619–1622 and references therein.
4.9. In vivo anticancer activity
[7] (a) L. Jigyu, M. Zhida, M. Niwa, Acta Chim. Sin. 46 (1988) 1053;
(b) S.K. Chattopadhyay, R.P. Sharma, Phytochemistry 39 (1995) 935–936.
[8] L.O. Zamir, J. Zhang, J.H. Wu, F. Sauriol, O. Mamer, Tetrahedron 55 (1999)
14323–14340.
Animal studies were conducted with prior approval of Institu-
tional Animal Ethics Committee, CDRI, Lucknow. Mammary tumors
were induced in virgin female Sprague Dawley rats with gastric
[9] S.J. Yang, J.M. Fang, Y.S. Cheng, Phytochemistry 43 (1996) 839–842.

K.P. Reddy et al. / European Journal of Medicinal Chemistry 44 (2009) 3947–3953
3953
[10] J.Y. Liang, K.S. Huang, A.A.L. Gunatilaka, Planta Med. 64 (1998) 187–188.
[11] K.V. Rao, J. Juchum, Phytochemistry 47 (1998) 1315–1324.
[12] K. Jun´ ichi, S. Hideyuki, Heterocycles 47 (1998) 1111–1134.
[13] N.T. Nguyen, A.H. Banskota, Y. Tezuka, T. Nobukawa, S. Kadota, Phytochemistry
64 (2003) 1141–1147.
[17] J.M. Khurana, P. Sharma, Bull. Chem. Soc. Jpn. 77 (2004) 549–552.
[18] M.A. Andersson, R. Epple, V.V. Fokin, K.B. Sharpless, Angew. Chem., Int. Ed. 41
(2002) 472–475.
[19] (a) I. Fenoglio, G.M. Nano, D.G. Vander Velde, G. Appendino, Tetrahedron Lett.
37 (1996) 3203–3206;
[14] J. Kobayashi, A. Ogiwara, H. Hosoyama, H. Shigemori, N. Yoshida, T. Sasaki,
Y.L.S. Iwasaki, M. Naito, T. Tsuruo, Tetrahedron 50 (1994) 7401–7416.
[15] J. Kobayashi, H. Hosoyama, X.X. Wang, H. Shigemori, Y. Koiso, S. Iwasaki,
(b) D.A. Sun, A. Nikolakakis, F. Sauriol, O. Mamer, L. Zamir, Bioorg. Med. Chem.
9 (2001) 1985–1992.
[20] H.D. Soule, J. Vazguez, A. Long, S. Albert, M. Brennan, J. Natl. Cancer Inst. 51
(1973) 1409–1416.
[21] K.V. Sashidhara, J.N. Rosaiah, A. Kumar, H.K. Bid, R. Konwar, N. Chattopadhyay,
Phytother. Res. 21 (2007) 1105–1108.
[22] C. Huggins, L.C. Grand, P.F. Brillantes, Nature 189 (1961) 204–207.
T. Sasaki, M. Naito, T. Tsuruo, Bioorg. Med. Chem. Lett.
393–398.
7 (1997)
[16] M. Botta, S. Armaroli, D. Castagnolo, G. Fontana, P. Perad, E. Bombardelli,
Bioorg. Med. Chem. Lett. 17 (2007) 1579–1583.