Syntheses and cytotoxicities of the analogues of the taxoid brevifoliol

Article abstract of DOI:10.1016/j.ejmech.2007.09.002

Seven novel brevifoliol analogues have been synthesized by coupling brevifoliol and 2-monosubstituted-4-phenyl-1,3-oxazolidine carboxylic acid after removal of the protecting group with acid treatment. Brevifoliol and its synthesized analogues were tested

Full text of DOI:10.1016/j.ejmech.2007.09.002

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1499e1505
http://www.elsevier.com/locate/ejmech
Original article
Syntheses and cytotoxicities of the analogues of the taxoid brevifoliol*
*
Sunil K. Chattopadhyay , Sarita Tripathi, Mahendra P. Darokar, Uzma Faridi,
Brijesh Sisodia, Shubhra Negi, J. Kotesh Kumar, Suman P.S. Khanuja
Central Institute of Medicinal and Aromatic Plants (CIMAP), P.O. CIMAP, Lucknow-226015, India
Received 10 May 2007; received in revised form 8 August 2007; accepted 6 September 2007
Available online 15 September 2007
Abstract
Seven novel brevifoliol analogues have been synthesized by coupling brevifoliol and 2-monosubstituted-4-phenyl-1,3-oxazolidine carboxylic
acid after removal of the protecting group with acid treatment. Brevifoliol and its synthesized analogues were tested for their cytotoxic activities
against four different human cancer cell lines, oral (KB), breast (MCF-7), colon (CaCO2) and liver (HepG-2) as determined by MTT assay. The
C-13 oxidized brevifoliol retained significant activity. Out of the seven analogues synthesized, C-13 oxidized brevifoliol-5-[N-tert-butoxycar-
bonyl amino-(20R,30S )-30-phenyl isoserine] analogue was interesting as it exhibited selective and potent cytotoxicity against liver cancer cell
line predominantly.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Brevifoliol analogues; Synthesis; Cytotoxicity; Selectivity
1. Introduction
assembly and cytotoxicity assays [6]. In both the assays, the
analogue showed little activity.
Brevifoliol was originally isolated from the leaves of Taxus
brevifolia [1]. Its correct structure was established by Georg
et al. and Appendino et al. [2,3] and was assigned
a 11(15 / 1)-abeo-taxa-4(20),11-diene skeleton. We have
also isolated brevifoliol from the leaves of the Himalayan
yew Taxus wallichiana with a yield of 0.012% and it was
found to be the most abundant taxoid in the leaves of the plant
[4]. In the process, we have also been able to isolate another
naturally occurring analogue of brevifoliol, 2-acetoxy brevifo-
liol with a yield of 0.002% [4]. Brevifoliol was found to have
significant cytotoxic activity against colon (CaCO2), oral
(KB), breast (MCF-7) and liver (HepG-2) cancer cell lines [5].
In a previous publication, Georg et al. have prepared only
one brevifoliol derivative 13-[N-benzoyl-(20R,30S )-30-phenyl
isoserinate] and it was tested for its activity in microtubule
In a later publication, Tremblay et al. have prepared and
characterized several acetyl, 2,2,2-trichloroethyl chlorofor-
mate (Troc), and bis-triethyl-silyl (Tes) derivatives of brevifo-
liol [7].
They have also described the preparation of brevifoliol de-
rivatives with a 3-phenyl lactate at C-5 and C-13 positions of
brevifoliol and also the same derivative brevifoliol-13-[N-ben-
zoyl-(20R,30S )-30-phenyl isoserinate] synthesized by Georg
et al. Different derivatives of 5-acetyl brevifoliol were also
synthesized via esterification with cinnamic acid, with both
S-(ꢀ) and R-(þ)-3-phenyllactic acid and with a [N-benzoyl-
(20R,30S)-30-phenyl isoserine at C-13.
The significant cytotoxic activity of brevifoliol [5] has
prompted us to synthesize analogues and derivatives of the
molecule which are not yet synthesized by others. The ratio-
nale behind synthesizing the analogues is based on making
the cytotoxicity of brevifoliol selective towards certain tumors
and increasing its cytotoxicity. As docetaxel (taxotere) is ap-
proximately two times more cytotoxic than paclitaxel (taxol)
[8], we have decided to synthesize the analogues of brevifoliol
*
CIMAP communication No.: 2007-51J.
* Corresponding author. Tel.: þ91 522 2359628; fax: þ91 522 2342666.
E-mail address: chattsk@yahoo.com (S.K. Chattopadhyay).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.09.002

1500
S.K. Chattopadhyay et al. / European Journal of Medicinal Chemistry 43 (2008) 1499e1505
and its closely related natural analogue 2-acetoxy brevifoliol
in which the hydroxyl groups will be selectively esterified
with the side chain [N-tert-butoxycarbonyl amino-(20R,30S )-
30-phenyl isoserine]. These types of analogues have not yet
been synthesized.
In this communication, we would like to report the selective
oxidation of C-13 allylic hydroxyl groups of brevifoliol 1 and
2-acetoxy brevifoliol 2 and esterification of the remaining hy-
droxyl group in the oxidized products of both the molecules
with the docetaxel side chain [N-tert-butoxycarbonyl amino-
(20R,30S )-30-phenyl isoserine]. Moreover, brevifoliol itself
was converted into three different analogues in which its C-
5, C-13 and both C-5 and C-13 hydroxyls were esterified
with the side chain [N-tert-butoxycarbonyl amino-(20R,30S )-
30-phenyl isoserine]. The cytotoxic activity of all the new mol-
ecules were evaluated and included in this publication.
used by Chordia and Kingston to oxidize the C-13 hydroxyl
group of 10-deacetyl baccatin-III [9]. We found that the reac-
tion of brevifoliol with MnO2 in acetone at room temperature
selectively oxidized the C-13 allylic hydroxyl group to pro-
duce the corresponding oxidized brevifoliol 3 cleanly. As
compared to brevifoliol, the 1H NMR of 3 was broad at
room temperature. However, the spectra were well resolved
at low temperature (ꢀ20 ꢁC) and all the signals were assigned.
In the low temperature (ꢀ20 ꢁC) 1H NMR spectrum of ox-
idized brevifoliol 3, the chemical shift of the H-10 underwent
a downfield shift and appeared at d 6.81 as compared to its
chemical shift at d 6.41 which indicated that the oxidation
of C-13 hydroxyl group of brevifoliol has taken place. 13C
NMR spectrum of the compound 3 also supported the pres-
ence of an enone type carbonyl (d 207.7). Moreover, in the
HMBC spectrum of 3, the C-18 methyl signal at d 2.1 was
correlated to the carbonyl signal at d 207.7 thus confirming
the oxidation of C-13 hydroxyl of brevifoliol. Direct coupling
of the oxidized brevifoliol with 2-monosubstituted-4-phenyl-
1,3-oxazolidine carboxylic acid [10] in presence of dicyclo-
hexylcarbodiimide (DCC), and dimethyl aminopyridine
(DMAP) in toluene gave the condensed product at room
temperature. Acid mediated cleavage of the oxazolidine ring
of the condensed product in methanol afforded the
desired product, oxidized brevifoliol-5-[N-tert-butoxycarbonyl
2. Results and discussion
2.1. Chemistry
We began our synthesis with the selective oxidation of bre-
vifoliol 1 (Scheme 1). Brevifoliol has three types of hydroxyl
groups-allylic at C-13, secondary at C-5 and tertiary at C-15.
Reaction of brevifoliol with MnO2 was tried as MnO2 was
OAc
19
BzO
OAc
19
BzO
OAc
18
OAc
18
12
12
9
10
9
10
7
7
4
11
8
6
5
8
11
6
5
(i)
13
13
O
HO
1
3
1
3
4
14
14
2
2
OH
OH
15
15
20
R
20
R
17
16
17
OH
16
OH
1. R = H, brevifoliol
3. R = H
2. R = OAc, 2-
4.R = OAc
acetoxybrevifoliol
(ii) + (iii)
OAc
BzO
OAc
18
19
12
9
10
7
11
8
3
6
5
NH-BOC
O
13
O
1
4
14
2'
2
1'
3'
O
Ph
15
20
OH
R
17
16
OH
5. R = H
6. R = OAc
BOC = tBuOCO
Scheme 1. Reagents and conditions: (i) MnO2, acetone (5 ml), 25 ꢁC, 30 h, 80%; (ii)
70%; (iii) p-TSA (1.1 equiv), MeOH (5 ml), 25 ꢁC, 2 h, 51%.
DCC (3 equiv), DMAP (0.2 equiv), toluene (3 ml), 25 ꢁC, 1 h,

S.K. Chattopadhyay et al. / European Journal of Medicinal Chemistry 43 (2008) 1499e1505
1501
amino-(20R,30S )-30-phenyl isoserine] 5 in 51% isolated yield.
Again, the NMR spectra of 5 were broad at room temperature
(23 ꢁC) which was well resolved at low temperature (ꢀ20 ꢁC).
Oxidation of 2-acetoxy brevifoliol 2 with MnO2 at room tem-
cell lines, oral (KB), breast (MCF-7), colon (CaCO2) and liver
(HepG-2) as determined by MTT assay [12,13] (Table 1).
From Table 1, it appears that although C-13 oxidized brevifo-
liol 3 was less active than brevifoliol, it still retained signifi-
cant cytotoxic activity against the above cancer cell line
except HepG-2 line. However, in case of C-13 oxidized 2-ace-
toxy brevifoliol 4, it became less cytotoxic as compared to its
precursor 2-acetoxy brevifoliol 2. Analogue 5 was interesting
as it exhibited selective and potent cytotoxicity against liver
cancer cell line predominantly. Similar selectivity was ob-
served with analogue 6 against oral cancer cell line with
more cytotoxicity. The three other analogues 7, 8, and 9 syn-
thesized from brevifoliol exhibited less cytotoxic activity as
compared to brevifoliol 1 as shown in Table 1. The only bre-
vifoliol analogue, 13-[N-benzoyl-(20R,30S )-30-phenyl isoseri-
nate] synthesized by Georg et al. showed cytotoxicity with
a ED50 value which was greater than 10 mM (¼8.23 mg/ml;
highest concentration tested) as compared to 27 nM for pacli-
taxel [6]. However, in our case, out of the three brevifoliol an-
alogues 7, 8 and 9, compound 9, the diesterified analogue of
brevifoliol exhibited selective and significant cytotoxicity
against oral cancer cell line (KB) (IC50 and IC90 are 2.5 and
30 mg/ml).
Thus, from the result of cytotoxicity data for brevifoliol an-
alogues 7, 8 and 9 synthesized by us, we can say that the re-
placement of the side chain of the paclitaxel with that of
docetaxel did not lead to the enhancement in cytotoxicity ex-
cept in case of compound 9 when compared with the data re-
ported by Georg et al. [6].
Thus, we have prepared and characterized C-13 oxidized
brevifoliol, C-13 oxidized 2-acetoxy brevifoliol and their con-
densed products with [N-tert-butoxycarbonyl amino-(20R,30S )-
30-phenyl isoserine]. Also, C-5, C-13 and C-5 and 13 analogues
of brevifoliol esterified with [N-tert-butoxycarbonyl amino-
(20R,30S )-30-phenyl isoserine] have been prepared. To our
knowledge, the above analogues of brevifoliol have been pre-
pared for the first time and some of them, e.g. 5 and 6,
possessed selective and significant cytotoxicity against liver
and oral cancer cell lines, respectively. The selective and sig-
nificant cytotoxicity of the oxidized brevifoliol 5 and 6 may be
1
perature gave 4. The diagnostic feature in the H NMR of 4
was the small coupling constant (3.2 Hz) between H-9 and
H-10 which suggested a chair/boat conformation for the B/C
ring [11]. Search in literature revealed that this molecule
has been isolated as a natural taxoid, taxuspinane B from
the stems of the plant Taxus cuspidata by Morita et al. [11].
2-Acetoxy brevifoliol 2 was converted into 6 following the se-
quence of reactions as described in Scheme 1. Georg et al. has
reported that condensation of brevifoliol with b-lactam (pre-
cursor of the taxol side chain) gave exclusively brevifoliol-
13-[N-benzoyl-(20R,30S )-30-phenylisoserinate] [6]. Reaction
of brevifoliol itself with 2-monosubstituted-4-phenyl-1,3-
oxazolidine carboxylic acid [10] in presence of dicyclohexyl-
carbodiimide (DCC), and dimethyl aminopyridine (DMAP) in
toluene gave three condensed products as expected. Cleavage
of the oxazolidine ring of the condensed products with acid
gave three products 7, 8 and 9 in a ratio of 6:3:1 in which
5, 13 and both 5 and 13 hydroxyl groups were esterified
with N-tert-butoxycarbonyl amino-(20R,30S )-30-phenyl isoser-
1
ine. As compared to the H NMR spectrum of the oxidized
brevifoliol 3, line broadening in the NMR spectra of 7 was
1
moderate and full assignment of the H and 13C NMR spectra
by 1D and 2D techniques (COSY, HSQC and HMBC) could
be achieved at room temperature.
In HMBC spectrum of 7, the signal at d 5.46 (s, H-5) was
correlated to the carbonyl signal at d 170.8 which indicated
that the C-5 hydroxyl group has been esterified with the side
chain [N-tert-butoxycarbonyl amino-(20R,30S )-30-phenyl iso-
serine]. The other two synthesized analogues 8 and 9 of brevi-
foliol displayed spectral data which were in agreement with
their assigned structures.
2.2. Cytotoxic activities
The brevifoliol analogues thus obtained were tested for
their cytotoxic activity against four different human cancer
Table 1
Cytotoxicity of brevifoliol and its analogues against human cancer cell lines by MTT assay (values in mg/ml)a
Compound KB
IC50 ꢂ s.d.
MCF-7
CaCO2
HepG2
IC90 ꢂ s.d.
IC50 ꢂ s.d.
IC90 ꢂ s.d.
IC50 ꢂ s.d.
IC90 ꢂ s.d.
IC50 ꢂ s.d.
IC90 ꢂ s.d.
1
0.0031 ꢂ 0.00047
0.316 ꢂ 0.0471
0.0078 ꢂ 0.00023
100 ꢂ 4.08
0.6 ꢂ 0.071
7.5 ꢂ 0.816
1.13 ꢂ 0.262
100 ꢂ 4.08
100 ꢂ 4.08
5.33 ꢂ 0.471
100 ꢂ 4.08
100 ꢂ 4.08
33.3 ꢂ 2.35
0.86 ꢂ 0.023
58.3 ꢂ 4.71
5.33 ꢂ 0.471
100 ꢂ 4.08
8.5 ꢂ 1.08
6.16 ꢂ 0.471 0.0025 ꢂ 0.00041
1.32 ꢂ 0.259 0.055 ꢂ 0.0071 1.13 ꢂ 0.262
2
91.6 ꢂ 2.35
22.6 ꢂ 2.054
100 ꢂ 4.08
43.33 ꢂ 4.71
100 ꢂ 4.08
55 ꢂ 4.08
6.16 ꢂ 1.027
0.008 ꢂ 0.0016
100 ꢂ 4.08
100 ꢂ 4.08
100 ꢂ 4.08
100 ꢂ 4.08
100 ꢂ 4.08
100 ꢂ 4.08
21 ꢂ 2.94
21 ꢂ 2.94
96.6 ꢂ 2.35
100 ꢂ 4.08
96.6 ꢂ 2.35
0.065 ꢂ 0.016
100 ꢂ 4.08
100 ꢂ 4.08
78.33 ꢂ 4.71
96.6 ꢂ 2.35
100 ꢂ 4.08
100 ꢂ 4.08
100 ꢂ 4.08
5.33 ꢂ 0.85
100 ꢂ 4.08
100 ꢂ 4.08
91.6 ꢂ 4.71
100 ꢂ 4.08
3
4
100 ꢂ 4.08
100 ꢂ 4.08
100 ꢂ 4.08
100 ꢂ 4.08
100 ꢂ 4.08
100 ꢂ 4.08
5
100 ꢂ 4.08
6
0.105 ꢂ 0.075
100 ꢂ 4.08
100 ꢂ 4.08
33.3 ꢂ 2.35
48.3 ꢂ 4.71
100 ꢂ 4.08
7
8
100 ꢂ 4.08
68.3 ꢂ 4.71
100 ꢂ 4.08
9
Paclitaxel
2.73 ꢂ 0.21
0.0013 ꢂ 0.0004
0.048 ꢂ 0.0012 0.008 ꢂ 0.0016
0.86 ꢂ 0.023 0.0078 ꢂ 0.00023 0.065 ꢂ 0.016 0.008 ꢂ 0.0016 1.13 ꢂ 0.262
s.d. is the standard deviation of three replicates.
a
Highest concentration tested for each compound was 100 mg/ml.

1502
S.K. Chattopadhyay et al. / European Journal of Medicinal Chemistry 43 (2008) 1499e1505
1
due to the conformational changes in the taxane rings which
helps in binding to the target of a particular cell (liver and
oral cancer cell lines).
1372, 1244 cmꢀ1. H NMR (CDCl3 at 23 ꢁC): 6.07 (1H, d,
J ¼ 9.2 Hz, 2-H), 2.77 (1H, d, J ¼ 9.2 Hz, 3-H), 4.75 (1H, br
t, 5-H), 1.18 (1H, m, 6a-H), 1.88 (1H, m, 6b-H), 4.84 (1H, t,
J ¼ 8.8 Hz, 7-H), 5.12 (1H, d, J ¼ 3.2 Hz, 9-H), 6.32 (1H, d,
J ¼ 3.2 Hz, 10-H), 2.47 (1H, d, J ¼ 18.6 Hz, 14 b-H), 2.77
(1H, d, J ¼ 18.6 Hz, 14a-H), 1.15 (3H, s, 16-H), 0.82 (3H,
s, 17-H), 2.00 (3H, s, 18-H), 1.84 (3H, s, 19-H), 4.79 (1H, br
s, 20a-H), 5.49 (1H, s, 20b-H), 1.96, 1.97, 1.98 (3 ꢃ 3H, s,
3 ꢃ OCOCH3), 8.01 (2H, d, J ¼ 7.4 Hz, o-Bz), 7.48 (2H, t,
J ¼ 7.4 Hz, m-Bz), 7.60 (1H, t, J ¼ 7.4 Hz, p-Bz). 13C NMR
(CDCl3 at 23 ꢁC): 59.1 (C-1), 69.9 (C-2), 45.9 (C-3), 147.1
(C-4), 68.6 (C-5), 36.1 (C-6), 70.5 (C-7), 44.8 (C-8), 75.7 (C-
9), 70.2 (C-10), 148.4 (C-11), 161.2 (C-12), 207.6 (C-13),
45.9 (C-14), 76.0 (C-15), 28.0 (C-16), 27.9 (C-17), 9.14 (C-
18), 14.2 (C-19), 111.7 (C-20), 21.6, 20.9, 20.8 (3 ꢃ CH3,
OCOCH3), 170.2, 170.1, 169.7 (3 ꢃ CO, OCOCH3), 167.9
(COeBz), 133.80, 133.7 (o-Bz), 130.1, 129.9 (m-Bz), 129.1
( p-Bz), 129.7 (C-1 Bz).
3. Experimental protocols
IR spectra, KBr pellets, 600e4000 cmꢀ1, were recorded
1
on PerkinElmer FTIR BX Spectrophotometer. H NMR and
13C NMR were recorded on a Bruker AVANCE 300 instru-
ment at 300 MHz (chemical shifts are expressed as d in parts
per million relative to TMS as internal standard. The follow-
ing abbreviations are used: singlet (s), broad singlet (br s),
doublet (d), broad doublet (br d), double doublet (dd), dis-
torted doublet (dist.d), triplet (t), broad triplet (br t), distorted
triplet (dt), multiplet (m). Mass spectra (electro spray ioniza-
tion in positive mode, ESIþ) were recorded on an API 3000
(Applied Biosystem) mass spectrometer; chromatographic
purifications were performed by silica gel 60e120 mesh
(Loba Chemie) column chromatography. All the synthesized
compounds gave satisfactory elemental analysis (carbon,
hydrogen).
3.1.2. General procedure for synthesis of 5e9
The 2-monosubstituted-4-phenyl-1,3-oxazolidine carbox-
ylic acid as mentioned in Schemes 1 and 2 was prepared by
following a literature procedure [9]. A mixture of oxazolidine
carboxylic acid (1 mmol), dicyclohexyldicarbodiimide (DCC,
1.01 mmol), compound 3 (0.33 mmol) and 4-dimethyl amino-
pyridine (DMAP, 0.1 mmol) in dry toluene (3 ml) were stirred
for 2 h. The reaction mixture was concentrated and filtered
with benzene (3 ꢃ 5 ml). The residue thus obtained was puri-
fied by column chromatography using a mixture of EtOA-
c:hexane (25:75) to obtain the condensed product. It was
dissolved in MeOH (5 ml) and treated with p-TSA (1.1 equiv)
at 25 ꢁC for 1 h. The reaction mixture was concentrated and
purified by preparative TLC (20 ꢃ 20 cm, mobile phase
C6H6:Me2CO (3:1)) to yield compound 5.
3.1. Synthesis
3.1.1. General procedure for synthesis of 3 and 4
To a solution of compound 1 (0.36 mmol) in acetone (5 ml)
was added MnO2 (1 g) and the mixture was stirred at 25 ꢁC for
40 h. The reaction was filtered over a bed of celite and the or-
ganic phase was concentrated at reduced pressure. The residue
thus obtained could not be induced to crystallization but solid-
ified from hexane:acetone mixture to give compound 3.
Similar procedure was repeated with compound 2 to get
compound 4.
Similar procedure was repeated with compound 4 to get
compound 6 and with compound 1 to get three compounds
7, 8 and 9.
3.1.1.1. Compound 3. Yield 80%, amorphous solid. Mass
(ESIþ) [M þ Na]þ: m/z 577. IR (KBr): 3449, 2975, 2934,
1
1722, 1373 cmꢀ1. H NMR (CDCl3 at ꢀ20 ꢁC): 2.6 (2H, m,
2-H), 2.63 (1H, m, 3-H), 4.33 (1H, br s, 5-H), 1.25 (1H, m,
6a-H), 2.17 (1H, m, 6b-H), 5.60 (1H, dd, J ¼ 5.0 and 10.5 Hz,
7-H), 6.20 (1H, d, J ¼ 10.8 Hz, 9-H), 6.81 (1H, d,
J ¼ 10.8 Hz, 10-H), 2.01 (1H, d, J ¼ 18.1 Hz, 14 b-H), 2.55
(1H, d, J ¼ 18.1 Hz, 14a-H), 0.96 (3H, s, 16-H), 0.88 (3H,
s, 17-H), 1.37 (3H, s, 18-H), 1.22 (3H, s, 19-H), 4.78 (1H, br
s, 20a-H), 5.14 (1H, br s, 20b-H), 1.75, 2.02 (2 ꢃ 3H, s,
OCOCH3), 7.84 (2H, d, J ¼ 7.5 Hz, o-Bz), 7.46 (2H, t,
J ¼ 7.5 Hz, m-Bz), 7.56 (1H, t, J ¼ 7.5 Hz, p-Bz). 13C NMR
(CDCl3 at ꢀ20 ꢁC): 59.0 (C-1), 27.8 (C-2), 37.5 (C-3), 147.1
(C-4), 68.6 (C-5), 36.1 (C-6), 69.9 (C-7), 45.9 (C-8), 77.0 (C-
9), 70.3 (C-10), 163.9 (C-11), 147.1 (C-12), 207.7 (C-13),
50.1 (C-14), 76.1 (C-15), 9.2 (C-16), 12.9 (C-17), 14.3 (C-18),
27.0 (C-19), 111.7 (C-20), 20.9, 21.6 (2 ꢃ CH3, OCOCH3),
170.2, 170.3 (2 ꢃ CO, OCOCH3), 163.9 (COeBz), 128.8
(o-Bz), 129.9 (m-Bz), 133.8 ( p-Bz), 129.6 (C-1 Bz).
3.1.2.1. Compound 5. Yield 70%, amorphous solid. Mass
(ESIþ) [M þ H]þ: m/z 840. IR (KBr): 3446, 2977, 1716,
1
1498, 1370, 1255 cmꢀ1. H NMR (CDCl3 at ꢀ20 ꢁC): 2.65
(1H, m, 2a-H), 1.75 (1H, m, 2b-H), 2.70 (1H, br s, 3-H), 5.48
(1H, br s, 5-H), 2.20 (1H, m, 6a-H), 1.35 (1H, m, 6b-H), 5.55
(1H, t, J ¼ 9.3 Hz, 7-H), 6.25 (1H, d, J ¼ 10.8 Hz, 9-H), 6.91
(1H, d, J ¼ 10.8 Hz, 10-H), 1.25 (1H, d, J ¼ 9.0 Hz, 14a-H),
2.43 (1H, d, J ¼ 9.0 Hz, 14b-H), 0.93 (3H, s, 16-H), 1.36
(3H, s, 17-H), 2.14 (3H, s, 18-H), 1.25 (3H, s, 19-H), 4.95
(1H, br s, 20a-H), 5.34 (1H, br s, 20b-H), 1.76, 2.08 (2 ꢃ 3H,
s, 2 ꢃ OCOCH3), 4.26 (1H, br s, 20-H), 5.12 (1H, d,
J ¼ 10 Hz, 30-H), 1.39 (9H, s, t-Bu), 7.87 (2H, d, J ¼ 7.5 Hz,
o-Bz), 7.55 (1H, t, J ¼ 7.5 Hz, p-Bz), 7.46 (2H, t, J ¼ 7.5 Hz,
13
m-Bz), 7.3e7.4 (5H, m, Ph). C NMR (CDCl3 at ꢀ20 ꢁC):
59 (C-1), 28.07 (C-2), 30.0 (C-3), 139.3 (C-4), 70.01 (C-5),
33.8 (C-6), 74.19 (C-7), 45.5 (C-8), 76.2 (C-9), 71.0 (C-10),
163.7 (C-11), 145.2 (C-12), 207.7 (C-13), 50.13 (C-14), 75.5
(C-15), 9.10, 13.10, 27.0, 26.4 (4 ꢃ CH3), 21.5, 21.0
(2 ꢃ CH3, OCOCH3), 115.0 (C-20), 169.8, 170.2 (2 ꢃ CO,
3.1.1.2. Compound 4. Yield 70%, amorphous solid. Mass
(ESIþ) [M þ Na]þ: m/z 635. IR (KBr): 3451, 2933, 1717,

S.K. Chattopadhyay et al. / European Journal of Medicinal Chemistry 43 (2008) 1499e1505
1503
OAc
19
BzO
OAc
18
12
9
10
7
4
11
8
3
6
5
13
HO
1
14
2
OH
15
20
17
16
OH
1
(ii) + (iii)
OAc
19
BzO
OAc
18
OAc
19
NH-BOC
BzO
O
OAc
12
18
9
10
17
7
4
NH-BOC
12
8
3
11
1
9
6
5
O
10
17
7
4
13
6
5
11
8
3
HO
13
14
O
1
2'
2
3'
1'
Ph
14
O
Ph
2
15
OH
15
OH
20
OH
16
20
OH
16
OH
8
7
OAc
19
BzO
OAc
18
NH-BOC
O
12
10
9
7
NH-BOC
11
8
6
5
O
2''
3''
1''
13
Ph
O
1
3
14
4
2'
2
3'
1'
OH
O
Ph
15
OH
20
16
17
OH
9
Scheme 2. Reagents and conditions: (i)
25 ꢁC, 2 h, 51%.
DCC (3 equiv), DMAP (0.2 equiv), toluene (3 ml), 25 ꢁC, 1 h, 70%; (ii) p-TSA (1.1 equiv), MeOH (5 ml),
OCOCH3), 171.6 (C-10, eCOe), 164.5 (CO of Bz), 155.6 (CO
of carbamate), 74.3 (C-20), 57.2 (C-30), 28.6 (CH3, t-Bu), 80.13
(Cet-Bu), 129.0 (C-1 Bz), 129.2 (C-2,6 Bz), 127.2 (C-3,5 Bz),
133.8 (C-4 Bz), 128.0 (o-Ph), 129.1 (m-Ph), 129.0 ( p-Ph),
139.3 (C-1 Ph).
7.61 (1H, dt, p-Bz), 7.49 (2H, dt, m-Bz), 7.38e7.27 (5H, m,
13
Ph). C NMR (CDCl3, 23 ꢁC): 68.6 (C-1), 69.8 (C-2), 44.7
(C-3), 148.4 (C-4), 75.9 (C-5), 42.2 (C-6), 73.9 (C-7), 44.6
(C-8), 76.9 (C-9), 70.8 (C-10), 148.0 (C-11), 161.5 (C-12),
207.2 (C-13), 44.65 (C-14), 75.9 (C-15), 29.3 (C-16), 28.1
(C-17), 9.6 (C-18), 14.2 (C-19), 115.7 (C-20), 21.8, 21.2,
20.9 (3 ꢃ CH3, OCOCH3), 169.7 (3 ꢃ CO, OCOCH3), 171.7
(C-10, eCOe), 165.5 (CO of Bz), 155.5 (CO of carbamate),
72.8 (C-20), 58.2 (C-30), 28.2 (CH3, t-Bu), 80.3 (Cet-Bu),
129.0 (C-1 Bz), 129.2 (C-2,6 Bz), 127.2 (C-3,5 Bz), 131.1
(C-4 Bz), 128.1 (o-Ph), 129.1 (m-Ph), 129.0 ( p-Ph), 133.9
(C-1 Ph).
3.1.2.2. Compound 6. Yield 60%, amorphous solid. Mass
(ESIþ) [M þ Na]þ: m/z 898. IR (KBr): 3449, 2970, 1742,
1718, 1498, 1370, 1240 cmꢀ1 1H NMR (CDCl3, 23 ꢁC):
.
6.08 (1H, d, J ¼ 9 Hz, 2-H), 2.82 (1H, d, J ¼ 9 Hz, 3-H),
5.29 (1H, br s, 5-H), 2.10 (1H, m, 6a-H), 1.81 (1H, s, 6b-
H), 5.68 (1H, dt, 7-H), 5.12 (1H, d, J ¼ 3.3 Hz, 9-H), 6.22
(1H, d, J ¼ 3.3 Hz, 10-H), 2.45 (1H, d, J ¼ 18.6 Hz, 14a-H),
2.75 (1H, d, J ¼ 18.6 Hz, 14b-H), 1.07 (3H, s, 16-H), 1.25
(3H, s, 17-H), 1.40 (3H, s, 18-H), 0.81 (3H, s, 19-H), 5.30
(1H, br s, 20b-H), 4.85 (1H, br s, 20a-H), 1.88, 1.95, 2.00
(3 ꢃ 3H, s, OCOCH3), 4.45 (1H, br s, 20-H), 5.30 (1H, br s,
30-H), 1.30 (9H, s, t-Bu), 7.87 (2H, d, J ¼ 7.8 Hz, o-Bz),
3.1.2.3. Compound 7. Yield 60%, amorphous solid. Mass
(ESIþ) [M þ Na]þ: m/z 842. IR (KBr): 3427, 2977, 2933,
1
1738, 1370, 1260 cmꢀ1. H NMR (CDCl3): 1.40 (1H, m, 2a-
H), 2.4 (1H, m, 2b-H), 2.75 (1H, d, J ¼ 8.4 Hz, 3-H), 5.46
(1H, br s, 5-H), 2.1 (1H, m, 6a-H), 1.21 (1H, m, 6b-H), 5.53

1504
S.K. Chattopadhyay et al. / European Journal of Medicinal Chemistry 43 (2008) 1499e1505
(1H, dt, 7-H), 6.0 (1H, d, J ¼ 10.5 Hz, 9-H), 6.60 (1H, d,
J ¼ 10.5 Hz, 10-H), 4.47 (1H, br t, 13-H), 1.25 (1H, m, 14a-
H), 2.42 (1H, m, 14b-H), 0.98 (3H, s, 16-H), 1.30 (3H, s, 17-
H), 2.13 (3H, s, 18-H), 0.88 (3H, s, 19-H), 5.27 (1H, br s,
20a-H), 4.89 (1H, br s, 20b-H), 1.73, 2.03 (2 ꢃ 3H, s,
2 ꢃ OCOCH3), 4.45 (1H, br s, 20-H), 5.20 (1H, br s, 30-H),
1.30 (9H, s, t-Bu), 7.84 (2H, d, J ¼ 7.3 Hz, o-Bz), 7.52 (1H, t,
J ¼ 7.3 Hz, p-Bz), 7.40 (2H, t, J ¼ 7.4 Hz, m-Bz), 7.2e7.3
(5H, m, Ph). 13C NMR (CDCl3): 63.7 (C-1), 29.6 (C-2), 39.6
(C-3), 151.4 (C-4), 75.8 (C-5), 34.2 (C-6), 70.2 (C-7), 45.2
(C-8), 77.5 (C-9), 71.2 (C-10), 139.7 (C-11), 145.6 (C-12),
77.6 (C-13), 47.5 (C-14), 75.3 (C-15), 13.3 (C-18), 12.1 (C-
19), 114.6 (C-20), 21.6, 21.0 (2 ꢃ CH3, OCOCH3), 170.2
(2 ꢃ CO, OCOCH3), 170.8 (C-10, eCOe), 75.3 (C-20), 60.7
(C-30), 155.99 (CO of carbamate), 28.7 (CH3, t-Bu), 80.2 (Ce
t-Bu), 164.5 (CO of Bz), 129.7 (C-1 Bz), 129.8 (C-2,6 Bz),
127.3 (C-3,5 Bz), 133.6 (C-4 Bz), 127.9 (o-Ph), 128.6 (m-Ph),
129.1 ( p-Ph), 139.65 (C-1 Ph).
(2 ꢃ 3H, s, 2 ꢃ OCOCH3), 4.35 (1H, m, 20-H), 4.84 (1H, m,
200-H) 5.12 (1H, m, 30-H), 1.34 (9H, s, t0-Bu), 1.36 (9H, s,
t00-Bu) 7.86 (2H, d, J ¼ 7.5 Hz, o-Bz), 7.55 (1H, t,
J ¼ 7.5 Hz, p-Bz), 7.43 (2H, t, J ¼ 7.5 Hz, m-Bz), 7.22e7.33
(2 ꢃ 5H, m, Ph0 þ Ph00). 13C NMR (CDCl3): 62.8 (C-1), 29.7
(C-2), 39.3 (C-3), 146.8 (C-4), 74.4 (C-5), 34.1 (C-6), 69.8
(C-7), 45.4 (C-8), 77.7 (C-9), 70.1 (C-10), 137.7 (C-11),
145.1 (C-12), 80.5 (C-13), 43.8 (C-14), 75.9 (C-15), 27.6
(C-16), 25.6 (C-17), 12.0 (C-18), 13.2 (C-19), 114.2 (C-20),
21.5, 20.9 (2 ꢃ CH3, OCOCH3), 170.2, 171.2 (2 ꢃ CO,
OCOCH3), 169.8 (C-10, eCOe), 173.3 (C-100, eCOe), 76.1
(C-20), 73.7 (C-200), 57.5 (C-30), 54.5 (C-300), 155.8 (CO of
carbamate0), 155.7 (CO of carbamate00), 28.7 (CH3, t0-Bu),
28.6 (CH3, t00-Bu), 81.8 (Cet0-Bu), 80.1 (Cet00-Bu), 164.5
(CO of Bz), 131.1 (C-1 Bz), 129.9 (C-2,6 Bz), 128.9 (C-3,5
Bz), 129.0 (C-4 Bz), 128.9 (o-Ph0), 128.7 (o-Ph00), 127.9
(m-Ph0), 127.3 (m-Ph00), 129.0 ( p-Ph0), 128.9 ( p-Ph00), 137.0
(C-1 Ph0), 137.0 (C-1 Ph00).
3.2. Biology
3.1.2.4. Compound 8. Yield 30%, amorphous solid. Mass
(ESIþ) [M þ Na]þ: m/z 842. IR (KBr): 3445, 2977, 2935,
1
1739, 1703, 1502, 1370, 1260 cmꢀ1. H NMR (CDCl3): 1.44
The human tumor cell lines KB, MCF-7, CaCO2, HepG-2
were obtained from the National Centre for Cell Sciences
(NCCS), Pune, India.
(br d, 1H, 2a-H), 2.44 (dd, J ¼ 5 and 14 Hz, 1H, 2b-H),
3.02 (d, J ¼ 9.0 Hz, 1H, 3-H), 4.32 (br s, 1H, 5-H), 2.03 (m,
1H, 6a-H), 1.76 (m, 1H, 6b-H), 5.73 (dd, J ¼ 11.0 and
5.2 Hz, 1H, 7-H), 5.96 (d, J ¼ 10.4 Hz, 1H, 9-H), 6.73 (d,
J ¼ 10.4 Hz, 1H, 10-H), 5.54 (dt, 1H, 13-H), 2.49 (m, 1H,
14b-H), 1.22 (m, 1H, 14a-H), 1.06 (s, 3H, 16-H), 1.31 (s,
3H, 17-H), 2.16 (s, 3H, 18-H), 0.87 (s, 3H, 19-H), 5.07 (br
s, 1H, 20a-H), 4.67 (br s, 1H, 20b-H), 1.76, 2.06 (2s,
2 ꢃ 3H, OCOCH3), 4.53 (br s, 1H, 20-H), 5.32 (d,
J ¼ 10.5 Hz, 1H, 30-H), 5.40 (br s, 1H, NH), 1.34 (s, 9H, t-
Bu), 7.87 (d, J ¼ 7.3 Hz, 2H, o-Bz), 7.55 (t, J ¼ 7.3 Hz, 1H,
p-Bz), 7.38 (t, J ¼ 7 Hz, 2H, m-Bz), 7.45e7.33 (m, 5H, Ph).
13C NMR (CDCl3): 64.8 (C-1), 29.2 (C-2), 36.1 (C-3),
151.0 (C-4), 72.6 (C-5), 36.1 (C-6), 70.6 (C-7), 45.6 (C-
8), 77.3 (C-9), 71.0 (C-10), 138 (C-11), 146 (C-12), 82.8
(C-13), 43.7 (C-14), 75.7 (C-15), 27.2 (C-16), 25.11
(C-17), 12.4 (C-18), 13.4 (C-19), 110.8 (C-20), 21.7,
21.0 (2 ꢃ CH3, OCOCH3), 170.3 (2 ꢃ OCOCH3), 173.1
(C-10), 72.6 (C-20), 55.88 (C-30), 28.59 (CH3, t-Bu), 80.6
(Cet-Bu), 155.63 (CO of carbamate), 164.6 (CO of Bz),
129.7 (C-1 Bz), 129.9 (C-2,6 Bz), 127.2 (C-3,5 Bz), 133.6
(C-4 Bz), 128.1 (o-Ph), 129.1 (m-Ph), 129.0 ( p-Ph), 139.7
(C-1 Ph).
3.2.1. Cytotoxic assay
IC50 values were determined and the cell survival was mea-
sured by using the MTT assay described by Mosman [13].
Briefly, 0.5 or 1 ꢃ 105 cells/ml cells at the exponential growth
phase were taken in a flat-bottomed 96-well polystyrene-
coated plate and were incubated for 24 h in CO2 incubator
at 5% CO2 and 37 ꢁC. Compound was added in concentrations
of 100, 10, 1, 0.1, 0.01, and 0.001 mg/ml medium. After 48 h
incubation, 10 ml/well MTT (stock solution 5 mg/ml PBS)
was added for 4 h and formazan crystals so formed were dis-
solved in 100 ml DMSO. The plates were read immediately in
a microplate reader (Spectramex, 190 Molecular Devices Inc.,
USA) operating at 570 nm. Wells with complete medium,
compound, and MTT, but without cells were used as blanks.
IC50 and IC90 values were expressed as micrograms of com-
pound concentration per milliliter that caused a 50% and
90% inhibition of growth compared with controls. Paclitaxel
was used as positive controls in every experiment.
References
[1] F. Balza, S. Tachibana, H. Barrios, G.H.N. Towers, Phytochemistry 30
(1991) 1613e1614.
3.1.2.5. Compound 9. Yield 10%, amorphous solid. Mass
(ESIþ) [M þ Na]þ: m/z 1105. IR (KBr): 3428, 2971, 2931,
[2] G.I. Georg, S.R. Gollapudi, G.L. Grunewald, C.W. Gunn, R.H. Himes,
B.K. Rao, X.Z. Liang, Y.W. Mirhom, L.A. Mitscher, D.G.V. Velde,
Q.M. Ye, Bioorg. Med. Chem. Lett. 3 (1993) 1345e1348.
[3] G. Appendino, L. Barboni, P. Gariboldi, E. Bombordelli, B. Gabetta,
D. Viterbo, J. Chem. Soc., Chem. Commun. (1993) 1587e1589.
[4] S.K. Chattopadhyay, G.C. Saha, M. Kulshrestha, R.P. Sharma, S. Kumar,
Indian. J. Chem. 35B (1996) 175e177.
1
1736, 1726, 1498, 1369, 1258 cmꢀ1. H NMR (CDCl3): 1.44
(1H, m, 2a-H), 2.39 (1H, m, 2b-H), 2.86 (1H, d, J ¼ 6.3 Hz,
3-H), 4.41 (1H, s, 5-H), 2.31 (1H, br s, 6a-H), 1.65 (1H, br
s, 6b-H), 5.52e5.66 (3H, m, 7 þ 13 þ 300-H), 6.13 (1H, d,
J ¼ 10.3 Hz, 9-H), 6.76 (1H, d, J ¼ 10.3 Hz, 10-H), 1.25
(1H, m, 14a-H), 2.42 (1H, m, 14b-H), 1.12 (3H, s, 16-H),
1.34 (3H, s, 17-H), 2.16 (3H, s, 18-H), 0.85 (3H, s, 19-H),
4.89 (1H, br s, 20a-H), 5.31 (1H, br s, 20b-H), 1.73, 2.07
[5] S.P.S. Khanuja, T.R. Santha Kumar, A. Garg, R.K. Misra, S.K. Chatto-
padhyay, S. Srivastva, A.S. Negi, United States Patent Publication No.
20040127561 A1, 2002.

S.K. Chattopadhyay et al. / European Journal of Medicinal Chemistry 43 (2008) 1499e1505
1505
[6] G.I. Georg, Z.S. Cheruvallath, D.V. Velde, Q.M. Ye, L.A. Mitscher,
R.H. Himes, Bioorg. Med. Chem. Lett. 3 (1993) 1349e1350.
[7] S. Tremblay, C. Soucy, N. Towers, P.J. Gunning, L. Breau, J. Nat. Prod.
67 (2004) 838e845.
[10] E. Didier, E. Fouque, I. Taillepied, A. Commercon, Tetrahedron Lett. 35
(1994) 2349e2352.
[11] H. Morita, A. Gonda, L. Wei, Y. Yamamura, K. Takeya, H. Itokawa,
J. Nat. Prod. 60 (1997) 390e392.
[8] L.R. Kelland, G. Abel, Cancer Chemother. Pharmacol. 30 (1992)
444e450.
[9] M.D. Chordia, D.G.I. Kingston, Tetrahedron 53 (1997) 5699e5710.
[12] A.C. Beekman, A.R.W. Barensten, H. Woerdenbag, W.V. Uden, N. Pras,
J. Nat. Prod. 60 (1997) 325e330.
[13] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.