Design and synthesis of de novo cytotoxic alkaloids by mimicking the bioactive conformation of paclitaxel

Article abstract of DOI:10.1016/j.bmc.2010.07.069

Novel paclitaxel-mimicking alkaloids were designed and synthesized based on a bioactive conformation of paclitaxel, that is, REDOR-Taxol. The alkaloid 2 bearing a 5-7-6 tricyclic scaffold mimics REDOR-Taxol best among the compounds designed and was found to be the most potent compound against several drug-sensitive and drug-resistant human cancer cell lines. MD simulation study on the paclitaxel mimics 1 and 2 as well as REDOR-Taxol bound to the 1JFF tubulin structure was quite informative to evaluate the level of mimicking. The MD simulation study clearly distinguishes the 5-6-6 and 5-7-6 tricyclic scaffolds, and also shows substantial difference in the conformational stability of the tubulin-bound structures between 2 and REDOR-Taxol. The latter may account for the large difference in potency, and provides critical information for possible improvement in the future design of paclitaxel mimics.

Full text of DOI:10.1016/j.bmc.2010.07.069

Bioorganic & Medicinal Chemistry 18 (2010) 7101–7112
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of de novo cytotoxic alkaloids by mimicking the
bioactive conformation of palitaxel
Liang Sun a, Jean M. Veith c, Paula Pera c, Ralph J. Bernacki c, Iwao Ojima a,b,
⇑
a Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794–3400, USA
b Institute of Chemical Biology and Drug Discovery, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, USA
c Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel paclitaxel-mimicking alkaloids were designed and synthesized based on a bioactive conformation
of paclitaxel, that is, REDOR-Taxol. The alkaloid 2 bearing a 5-7-6 tricyclic scaffold mimics REDOR-Taxol
best among the compounds designed and was found to be the most potent compound against several
drug-sensitive and drug-resistant human cancer cell lines. MD simulation study on the paclitaxel mimics
1 and 2 as well as REDOR-Taxol bound to the 1JFF tubulin structure was quite informative to evaluate the
level of mimicking. The MD simulation study clearly distinguishes the 5-6-6 and 5-7-6 tricyclic scaffolds,
and also shows substantial difference in the conformational stability of the tubulin-bound structures
between 2 and REDOR-Taxol. The latter may account for the large difference in potency, and provides
critical information for possible improvement in the future design of paclitaxel mimics.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 14 June 2010
Revised 27 July 2010
Accepted 30 July 2010
Available online 6 August 2010
Keywords:
Paclitaxel-mimics
Anticancer
Cytotoxicity
Alkaloid
Molecular-modeling
REDORE-Taxol
Tubulin
R2O
O
OH
1. Introduction
O
R1
NH
O
Cancer is the first leading cause of death for people under the
age of 85 in the US.1,2 Paclitaxel (TaxolÒ) and docetaxel (TaxotereÒ)
currently serve as the most widely used drugs in the fight against
cancer among a variety of chemotherapeutic agents (Fig. 1).3,4 The
drugs bind to the b-tubulin subunit, accelerate the polymerization
of tubulin and stabilize the resultant microtubules, which leads to
apoptosis through cell-signaling cascade.5,6 New generation tax-
oids with much higher activities have been developed through
structure-activity relationship (SAR) studies.7–11
4
13
1
2
2'
O
3'
O
H
14
HO
O
O
O
OH
O
R1 = Ph, R2 = Ac: paclitaxel
1 = OtBu, R2 = H: docetaxel
R
Figure 1. Paclitaxel and docetaxel.
Since the paclitaxel structure is highly complex, especially its
baccatin component, it would be exciting if we can design and de-
velop paclitaxel mimics with much simpler structure, retaining po-
tent anticancer activity.12,13 Thus, some pioneering efforts along
this line have been made by a couple of research groups. In 2004,
Ojima and coworkers reported four paclitaxel mimics with an ind-
olizidine alkaloid scaffold based on their pharmacophore model
(Fig. 2).14 Two hydroxyl groups were designed to mimic the C2
and C13 hydroxyl groups (atom–atom distance and dihedral angle)
of the baccatin III skeleton. Their mimics showed substantially re-
polymerization at the concentrations examined.14 In 2005, Génard,
Gérrite and coworkers synthesized eight steroidal compounds
bearing the phenylisoerine and benzoate side-chains to mimic
the ‘‘T-form” docetaxel (Fig. 2).15 Their mimics only showed four
orders of magnitude lower activity than docetaxel and did not
show any inhibitory activity for microtubule disassembly.15 Unex-
pectedly, however, two compounds showed inhibitory activity for
microtubule assembly.15 In 2006, Kingston and coworkers reported
five macrocyclic paclitaxel-mimics (Fig. 2) based on a highly potent
conformationally restrained macrocyclic paclitaxel analog, which
was designed to mimic the T-Taxol structure.16,17 Their mimics
duced but still good cytotoxicity (IC50 = single–double digit
lM)
against human breast cancer cell lines, but did not promote tubulin
showed fairly good cytotoxicity (IC50 = 11–18 lM) against a human
ovarian cancer cell line A2780 as well as weak, but recognizable
activity in promoting tubulin polymerization and stabilizing
⇑
Corresponding author. Tel.: +1 631 632 1339; fax: +1 631 632 7942.
E-mail address: iojima@notes.cc.sunysb.edu (I. Ojima).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.069

7102
L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
O
O
O
O
O
O
O
t-Boc
( )
n
Ph
NH
O
NH
O
N
H
N
H
(
)
n
N
H
TIPSO
N
H
HN
O
Ph
O
O
OH
OH
OH
O
O
O
OBz
4a: n = 0
4b: n = 1
OBz
O
1: n = 0
2: n = 1
O
OAc
O
O
MeO
A
B
OAc
O
MeO
Ojima's mimics
Ph
NH
O
R1
O
N
H
R2
( )n
NH
TIPSO
HO
O
O
OH
t-Boc
H
O
OBz
PhCONH
NH
O
HO
BzO
O
5a: n = 0
5b: n = 1
X'
O
Ph
O
OBz
3
OAc
H
O
O
OH
Kingston's mimics C
Roussi's mimics D
M
Br
NBoc
CHO
Figure 2. Previously reported paclixel mimics.
+
n
OR
n
OR
TIPSO
H
n = 0, 1
8
6
7
M = Li, MgBr, ZnCl
microtubules.16,17 We report here the design, synthesis and biolog-
ical evaluation of novel paclitaxel mimics, bearing tricyclic alkaloid
scaffolds, mimicking the REDOR-Taxol13,18,19 structure.
Scheme 1. Retrosynthesis of paclitaxel-mimicking alkaloids 1–3.
3. Synthesis of paclitaxel-mimicking alkaloids
2. Design and synthesis of novel paclitaxel mimics
As Scheme 1 shows, the retrosynthetic analysis indicates that (i)
compounds 1 and 2 can be obtained by introducing the phenyliso-
serine moiety to the tricyclic alkaloid scaffold 4, and (ii) compound
3 can be synthesized by introducing two olefinic side chains to 4
followed by ring-closing metathesis (RCM). Tricyclic alkaloid scaf-
fold 4 can be prepared by the coupling reaction of lithium or Grig-
nard species 7 with protected enantiopure prolinal 6, followed by
lactamization and acylations.
Preliminary study indicated that the coupling reaction of 7 with
6 was very sensitive to the bulkiness of the groups at the 2 and 6
positions of 7, and only small groups were tolerated in this cou-
pling. After screening of various 2,6-substituents, bromobenzene
8a with a vinyl group serving as a hydroxymethyl synthon was se-
lected for the synthesis of 4a via 5a (n = 0) (Scheme 2). An arylli-
thium species 7a (n = 0; R = Li) was generated by treating 8a
(n = 0; R = H) with 2.2 equivalents of n-BuLi. The coupling of 7a
with 6 in the presence of HMPA afforded the desired product 9a
in 81% yield, and the subsequent acetylation of hydroxyl groups
gave 10a in 97% yield. Since the diastereomers of 9a and 10a were
not separable by HPLC analysis, the determination of diastereomer
ratio (d.r.) had to wait after lactamization. The vinyl moiety of 10a
was subjected to ozonolysis, oxidization, esterification, N-depro-
tection and lactamization to afford the desired 5-6-6 tricyclic
We set out to design novel paclitaxel mimics through molecular
modeling analysis of tricyclic alkaloid skeletons by adding an acet-
oxymethylbenzene moiety to the previously reported indolizidine
scaffold, mentioned above. The acetoxymethyl moiety was in-
tended to mimic the acetoxy group of paclitaxel at the C4 position.
The molecular design and analysis of the novel paclitaxel mimics
were carried out using the REDOR-Taxol structure,13,18,19 which
was proposed based on the analysis of the tubulin-bound bioactive
conformation of paclitaxel. The REDOR-Taxol structure has been
successfully used for the design and synthesis of highly active con-
formationally rigidified macrocyclic taxoids.13,18
First, the designed tricyclic alkaloids with an N-benzoylphenyl-
isoserine side chain were analyzed by molecular mechanics (MM)
energy-minimization (InsightII 2000) at the binding site of paclit-
axel in b-tubulin (1TUB20). The binding structures were compared
with the REDOR-Taxol structure and the ones showing good over-
lays were selected. Mimics 1 and 2 have three ester groups in the
tricyclic ring systems to mimic the C2, C4 and C13 groups of the
baccatin III skeleton. Macrocyclic mimic 3 was also designed to
rigidify the N-benzoylphenylisoserine and acetoxy moieties. These
three mimics showed good overlay with the REDOR-Taxol struc-
ture in the 1TUB b-tubulin, as illustrated in Fig. 3.
O
O
Ph
NH
O
(
)
n
N
H
Ph
O
OH
OBz
1: n = 0
2: n = 1
OAc
O
O
Ph
NH
O
N
H
O
OH
O
OBz
O
His229
His229
His229
3
O
Figure 3. Overlays of 1 (cyan) (a), 2 (yellow) (b), 3 (magenta) (c), and REDOR-Taxol (green) in the 1TUB b-tubulin.

L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
7103
was obtained in good yield via 10c, using the same 6-step protocol
as that for 9a. The d.r. was 1:1. The coupling reaction using the
Grignard species generated from 8c afforded 9c in 74% yield, which
Br
NBoc
n
R1
TIPSO
6
a-e
n
was converted to 11c with a 3:2 d.r. in favor of the desirable 11c-
(entry 6).
a
f
OH
R1
8
9
The 5-7-6 tricyclic scaffold 11d was synthesized in a similar
manner from 8d (n = 1, R1 = CH2O-MOM) (Scheme 2). The coupling
of the aryllithium species generated from 8d with 6 gave 9c in 74%
yield. The subsequent acetylation afforded 10d in 96% yield, which
was converted to the 5-7-6 tricylclic scaffold 11d in 66% overall
yield through the same 5-step protocol as that for 10a (Scheme 2).
The d.r. was substantially increased to 10:1, but in favor of 11d-b.
Since this stereochemistry can be inverted through the Mitsunobu
reaction, further optimization of the coupling conditions was not
pursued.
8a: n = 0, R1 = CH2OH
9a: n = 0, R1 = CH2OH (81%)
9b: n = 0, R1 = CH2OMOM
9c: n = 0, R1 = CH=CH2 (74%)
9d: n = 1, R1 = CH2OMOM
8b: n = 0, R1 = CH2OMOM
8c: n = 0, R1 = CH=CH2
8d: n = 1, R1 = CH2OMOM
g
O
n
N
h-k
l
NBoc
n
TIPSO
5
TIPSO
H
AcO
OAc
10
R1
11
R1
Enantiopure tricyclic scaffold 11a-
pling-ready 5-6-6 tricyclic scaffolds 17a and 17b as illustrated in
Scheme 3. Basic hydrolysis of the two acetate groups of 11a- gave
a was converted to the cou-
11a: n =0, R1 = CH2OAc (50%)
11b: n =0, R1 = CH2OMOM (50%)
11c: n =0, R1 = COOMe (63%)
11d: n =1, R1 = CH2OMOM (66%)
10a: n = 0, R1 = CH2OAc (97%)
a
10b: n = 0, R1 = CH2OMOM( 70% for 2 steps)
10c: n = 0, R1 = CH=CH2 (98%)
12 in high yield, and the subsequent selective protection of the pri-
mary alcohol of 12 with TBDMSCl in the presence of imidazole pro-
ceeded with very high selectivity, affording 13 in 99% yield.26
Acylation of the secondary alcohol of 13 using 6 equivalents of
benzoyl chloride gave 14 in quantitative yield. Acidic deprotection
of TBDMS group afforded 15 in 95% yield.27 Acylation of the pri-
mary alcohol of 15 with acetic anhydride gave 16a and the subse-
quent deprotection of the TIPS group with HF/pyridine afforded
17a quantitatively. In the same manner, the acylation of 15 with
4-pentenoyl chloride, Et3N, DMAP followed by deprotection with
HF/pyridine gave 17b in quantitative yield.
10d: n = 1, R1 = CH2OMOM (70% for 2 steps)
Scheme 2. Synthesis of tricyclic alkaloid scaffold 11. Reagents and conditions: (a)–
(f) see Table 1; (g) Ac2O (1.5–3.0 equiv), Et3N (2.5–5.0 equiv), DMAP (0.2 equiv),
CH2Cl2, 0 °C to rt, overnight; (h) O3, CH2Cl2, ꢀ78 °C; Me2S, rt, 3 h; (i) NaClO2
(8 equiv), NaH2PO4 (8 equiv), acetone, H2O, rt, 40 min; (j) CH2N2, ether, rt, 10 min;
(k) CF3COOH, anisole (0.3 equiv), CH2Cl2, 0 °C to rt, 1 h; (l) NaHCO3, H2O, EtOAc, rt,
overnight.
scaffold 11a in 50% yield for 5 steps. Diastereomers, 11a-
a and
11a-b, were separated by column chromatography. It was found
that the d.r. was 2:1 with the undesirable diastereomer 11a-b as
the major product (Table 1, entry 1) although the Garner mod-
For the synthesis of enantiopure 5-7-6 tricyclic scaffold 17c,
11d-b was deacetylated to give 18 first, and then, 18 was subjected
el14,21 predicted that 11a-
a should be the major product. The ste-
O
O
reochemistry of the acetoxy group in 11a-
a as well as 11a-b was
N
H
N
H
a
unambiguously assigned by NOE analysis.
TIPSO
TIPSO
In the hope of possible improvement of diastereoselectivity in
the coupling reaction of 8 with 6, 8b and 8c were employed under
different coupling conditions (Scheme 2). Results are summarized
OAc
OH
OAc
OH
11a-α
12
b
in Table 1. Bromo(vinyl)benzene 8b with
a MOM-protected
hydroxymethyl group reacted with 6 to give 10b via 9b in good
yield (70% for 2 steps), and 10b was subjected to the same protocol
as that for 10a to afford 11b in 50% overall yield for five steps.22
The d.r. was found to be 3:4 in favor of 11b-b (entry 2). Next, 8b
was successfully converted to potentially more selective Grignard
species (entry 3) and zinc species (entry 4) by standard proce-
dures.23,24 However, the attempted coupling reaction did not yield
any desired product.
2,6-Divinylbromobenzene 8c, bearing no oxygen to coordinate
to lithium, was converted to the corresponding aryllithium spe-
cies25 and subjected to the coupling reaction with 6 to afford the
product 9c in 74% yield. The desired product 11c (R1 = CO2Me)
O
O
N
N
H
c
TIPSO
TIPSO
TIPSO
HO
H
OBz
d
OH
O
OTBDMS
13
OTBDMS
14
15
O
N
H
e
N
H
TIPSO
OBz
g, f
OBz
16a
OH
OAc
f
Table 1
O
Diastereoselectivity of the coupling of 8 with 6
O
N
H
N
Entry
Substrate
Conditions
11-a:11-b
HO
1
2
3
4
5
6
7
8a
8b
8b
8b
8c
8c
8d
a, b, f
c, b, f
d, f
c, e, b, f
c, b, f
d, f
1:2
3:4
NA
NA
1:1
3:2
OBz
H
O
O
OBz
OAc
17b
17a
Scheme 3. Synthesis of tricyclic scaffolds 17a and 17b. Reagents and conditions: (a)
K2CO3 (2.5 equiv), MeOH, H2O, rt, 30 min (84%); (b) TBDMSCl (1.5 equiv), Imidazole
(4.0 equiv), DMF, rt, 30 min (99%); (c) BzCl (6.0 equiv), Et3N (8.0 equiv), DMAP
(0.05 equiv), CH2Cl2, rt, overnight (100%); (d) 0.1 N HCl, EtOH, rt, 2.5 h (95%); (e)
Ac2O (2.0 equiv), Et3N (4.0 equiv), DMAP (0.03 equiv), CH2Cl2, rt, overnight (100%);
(f) HF/Py, CH3CN, Py, rt, overnight (100%); (g) 4-pentenoyl chloride (2 equiv), Et3N
(4 equiv), DMAP (0.03 equiv), CH2Cl2, rt, 1.5 h (100%).
c, b, f
1:10
Reagents and conditions: (a) n-BuLi (2.2 equiv), THF, ꢀ78 °C, 1 h; (b) HMPA, ꢀ78 °C,
1 h (c) n-BuLi (1.1 equiv), THF, ꢀ78 °C, 1 h; (d) Mg (1.2 equiv), BrCH2CH2Br, THF,
reflux, 2 h; (e) ZnCl2 (0.5 equiv), THF, 0 °C, 1 h; (f) 6 (1.0 equiv), ꢀ78 °C to rt,
overnight. NA = not applicable.

7104
L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
O
to the Mitsunobu reaction with benzoic acid in the presence of
DIAD and PPh3 (Scheme 4). The conversion of this reaction was
only 35% in refluxing THF for 3 days. Nevertheless, the desired
O
C6H4OMe-4
O
n
N
H
Ph
HO
N
+
a-benzoate 19 was obtained in 63% yield (based on 35% conver-
OBz
COOH
sion) for 2 steps, after deprotection of the MOM group. Acetylation
and the subsequent deprotection of the TIPS group with HF/pyri-
dine gave coupling-ready 17c in high yield. The inversion of config-
uration at the chiral center bearing the benzoate moiety was
confirmed by NOE analysis.
Tricyclic scaffolds 17a and 17c were coupled with oxazolidine
acid 20a (R2 = H), which was prepared by the literature meth-
od,28,29 in the presence of EDC and DMAP in CH2Cl2 to give 21a
and 21b. Deprotection of 21a and 21b with p-toluenesulfonic acid
(p-TsOH) in methanol afforded the designed paclitaxel-mimicking
alkaloids 1 and 2 in good yields (Scheme 5).
R2
OR
20a: R2 = H
17a: n = 0, R = Ac
a
17b: n = 0, R = 4-pentenoyl
17c: n = 1, R = Ac
20b: R2 = allyloxy
O
C6H4OMe-4
O
O
Ph
N
n
N
H
O
O
R2
OBz
OR
For the synthesis of the macrocyclic paclitaxel-mimicking alka-
loid 3, 20b was coupled with 17b, followed by deprotection to give
the final precursor, diene 22 in high yield in the same manner as
that described above. Finally, 22 was subjected to ring-closing
metathesis (RCM) using the first-generation Grubbs catalyst. The
reaction was very slow and not completed after 2 days in refluxing
dichloromethane. Nevertheless, the designed paclitaxel mimic 3
was obtained in 60% yield.
In order to examine the difference between the tricyclic alkaloid
scaffolds and the bicyclic alkaloid scaffold that we reported previ-
ously,14 paclitaxel mimic 24 was also synthesized through cou-
pling of 2314 with oxazolidine carboxylic acid 20a in the same
manner as that described above (Scheme 6).
21a: n = 0, R = Ac, R2 = H (82%)
21b: n = 0, R = 4-pentenoyl, R2 = allyloxy (96%)
21c: n = 1, R = Ac, R2 = H
b
O
O
Ph
NH
O
n
N
H
O
2OH
R
OBz
OR
1: n = 0, R = Ac, R2 = H (75%)
22: n = 0, R = 4-pentenoyl, R2 = allyloxy (82%)
n = 1, R = Ac, R2 = H (70% for 2 steps)
2:
c
for 22
O
4. Biological activity of novel paclitaxel mimics
O
Ph
NH
O
O
Novel paclitaxel mimics, thus obtained, were evaluated for their
cytotoxicities against drug-sensitive and drug-resistant human can-
cer cell lines and compared with those of previously reported paclit-
axel mimics. Results are summarized in Table 2. As Table 2 shows,
paclitaxel-mimic 2 bearing the 5-7-6 tricyclic scaffold is the most
potent among the novel paclitaxel-mimicking alkaloids synthe-
sized, exhibiting a single micromolar IC50 values against drug-sensi-
tive human breast cancer cell lines, MCF7-S, LCC6-WT, human
ovarian cancer cell line, A2780, and human pancreatic cancer cell
line, CFPAC-1 in spite of its drastically simplified structure (entry
6). The macrocyclic mimic 3, bearing a 5-6-6 tricyclic scaffold, is
the least potent among the newly synthesized paclitaxel-mimicking
N
H
OH
O
OBz
O
O
3 (60%)
Scheme 5. Synthesis of paclitaxel mimics 1-3. Reagents and conditions: (a) 20
(1.5 equiv), EDC (2 equiv), DMAP (0.5 equiv), CH2Cl2, rt, overnight; (b) p-TSA
(0.2 equiv), MeOH, rt, 10 h; (c) Cl2Ru(@CHPh)(PCy3)2 (0.2 equiv), CH2Cl2, reflux,
2 days.
O
O
O
Ph
NH
O
O
N
H
O
N
H
HO
Ph
O
N
H
N
H
a, b
a
OH
TIPSO
TIPSO
O
O
O
O
OH
18
OAc
11d-β
MeO
23
MeO
OMOM
OMOM
TIPSO
O
b, c
24
N
H
Scheme 6. Reagents and conditions: (a) 20a (1.5 equiv), EDC (2 equiv), DMAP
(0.5 equiv), CH2Cl2, rt, overnight; (b) p-TSA (0.2 equiv), MeOH, rt, 10 h, (76% for two
steps).
OBz
d
19
OH
O
O
e
N
H
N
alkaloids. Nevertheless, mimic 3 shows IC50 value (14.8
lM),
HO
TIPSO
comparable to Kingston’s mimics (IC50 = 11.3–20
l
M)14,17 against
H
OBz
OBz
16c
A2780 ovarian cancer cell line (entry 7). The polycyclic alkaloid
scaffold structures have critical effect on the potency of the mimics
(entries 5, 6 and 8), that is, the potency increases in the order,
24 (5-6 bicyclic) < 1 (5-6-6 tricyclic) < 2 (5-7-6 tricyclic), wherein
2 is substantially more potent than 1. Mimics 1 and 2 also possess
an acetoxy group, mimicking the acetoxy group at the C4 position
of paclitaxel, which might be contributing to better potency than
17c
OAc
OAc
Scheme 4. Synthesis of tricyclic scaffold 17c. Reagents and conditions: (a) K2CO3
(2.5 equiv), MeOH, H2O, rt, 30 min (81%); (b) DIAD (1.1 equiv), PhCOOH (1.2 equiv),
PPh3 (1.1 equiv), THF, reflux, 3 days; (c) anisole, CF3COOH, CH2Cl2, rt, 2 h (63% for 2
steps); (d) Ac2O (2 equiv), Et3N (3 equiv), DMAP (0.2 equiv), CH2Cl2, 0 °C-rt,
overnight (88%); (e) HF/Py, CH3CN, Py, rt, overnight (86%).

L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
7105
Table 2
In vitro cytotoxicities of paclitaxel mimics (IC50, l
M)a
Entry
Compounds
MCF7-Sb
NCI/ADRc
LCC6-WTd
LCC6-MDRe
A2780f
CFPAC-1g
1
2
3
4
5
6
7
8
Paclitaxel
0.0020 0.0002
5.70 0.26
8.10 0.22
N.A.
39.6 4.0
8.31 0.75
>100
0.410 0.045
8.70 0.18
13.0 0.6
N.A.
>100
41.5 4.5
>100
0.0030 0.0003
6.68 0.17
10.5 0.8
N.A.
36.3 3.5
4.93 0.39
>100
0.379 0.022
10.00 0.56
>20
N.A.
>100
16.8 1.9
>100
>100
0.020 0.002
N.D.
N.D.
10.9 1.5–20
7.80 0.13
3.81 0.33
14.8 1.5
N.D.
0.047 0.007
N.D.
N.D.
N.A.
>100
6.16 0.71
>100
>100
Mimic Ah
Mimic Bh
Mimic Ci
2
1
2
3
24
45.9 0.40
>100
42.8 0.26
a
b
c
d
e
f
Concentration of compound which inhibits 50% (IC50
MCF7-S: human breast carcinoma cell line (Pgpꢀ).
, lM) of the growth of human tumor cell line after 72 h drug exposure.
NCI/ADR: multi-drug resistant human ovarian carcinoma cell line (Pgp+).
LCC6-WT: human breast carcinoma cell line (Pgpꢀ).
LCC6-MDR: mdr1 transduced cell line (Pgp+).
A2780: human ovarian carcinoma cell line.
CFPAC-1: human pancreatic carcinoma cell line.
Ref. 14.
g
h
i
Ref. 17.
24. Based on the overlay, 2 is better mimicking paclitaxel than 1 for
this acetoxyl group.
(MD) studies on the mimics 1 and 2 in comparison with the RE-
DOR-Taxol structure in the 1JFF tubulin. The overlay of the energy
minimized structures of 1 and 2 in the 1JFF tubulin, using the
InsightII 2000 program (CVFF force field), with the REDOR-Taxol-
1JFF structure is shown in Figure 4.
The results also show clear difference between N-benzoylphe-
nylisoserine, that is, paclitaxel’s C13 side chain, and N-t-Boc-3-
(2-methylprop-2-enyl)isoserine, a C13 side chain of second- and
third-generation taxoids,10 with regard to their effect on the po-
tency of paclitaxel mimics (entries 2 and 8), that is, the potencies
of mimic A is one order of magnitude higher than those of 24,
wherein the only difference is the structure of the isoserine side
chain.
The resistance factor, that is, R-factor = IC50 (drug-resistant cell
line)/IC50 (drug-sensitive cell line), is an excellent indicator for the
sensitivity of cytotoxic agents against MDR phenotype (P-glyco-
protein overexpression, Pgp+). The R-factors of paclitaxel for the
MCF-7 versus NCI/ADR and LCC6-WT versus LCC6-ADR are 205
and 126, respectively (entry 1). In contrast, the R-factors of 2 for
the same pair of cell lines are 5.0 and 3.4, respectively (entry 6),
whereas those of A are 1.5 for both pairs of cell lines (entry 2).
Accordingly, mimics 2 and A are significantly less affected by the
P-glycoprotein efflux pump and the use of non-aromatic isoserine
side chain is found to be beneficial to further increase the potency
against MDR cancer cell lines.
The critical H-bond between the phenylisoserine moiety’s C20–
OH of 1 (1.8 Å) or 2 (2.1 Å) and His229 was very stable during the
energy minimization. As Figure 5 shows, the three structures have
very good matching for the 30-phenyl, 30N-benzoyl, and 2-benzoate
groups, as well as even the 4-acetoxyl group, although the oxetane
and the northern part of the baccatin structure are missing. Never-
theless, 1 and 2 only exhibit much lower potency than paclitaxel.
To obtain some insight into this observation, we carried out the
MD simulations of 1 and 2 in 1JFF using the Macromodel program
(MMFF94 force field33) and compared their conformational stabil-
ity with that of REDOR-Taxol-1JFF. In the MD simulations, all
atoms farther than 10 Å from the binding site were fixed, following
our previously reported protocol,19 and the stability of the C20–
OHꢁ ꢁ ꢁN(His229) H-bond was monitored during the whole simula-
tion in each case. The overlays of 200 snapshots for each (sampled
every 0.25 ps) of the conformations of 1, 2 and paclitaxel are
shown in Figure 5.
For the binding of mimics 2 to microtubules, a preliminary
study has been done through competitive displacement of Flu-
tax-230 using TR-NOESY analysis.31 The critical concentration of
tubulin required for microtubule assembly was determined to be
3.7 lM, while DMSO (vehicle) was 3.3 lM. This might suggest that
mimic 2 inhibits tubulin polymerization through low affinity inter-
action. Due to low water solubility of mimic 2, accurate binding
constant has not been determined. Nevertheless, the result ob-
served for Flutax-2 displacement indicates that the binding con-
stant (Kb) would be in 50–100
activity was also observed for
l
M range. Unexpected inhibitory
T-Taxol mimic (mimic D,
a
Fig. 2).15 These results may imply that there are compounds, which
interact with the paclitaxel binding site in b-tubulin, but destabi-
lize tubulin nucleates/oligomers. The study on the binding ability
of mimic 2 to tubulin will be reported elsewhere.
5. Molecular modeling analysis
When we first designed paclitaxel mimics 1, 2 and 3, the RE-
DOR-Taxol structure in the 1TUB20 tubulin was employed, as
shown in Figure 3. Since then, the REDOR-Taxol structure was re-
fined using the higher-resolution 1JFF32 b-tubulin.18,19 Accordingly,
we performed molecular modeling as well as molecular dynamics
Figure 4. Overlay of paclitaxel (REDOR-Taxol-1JFF) (green), 1 (cyan) and 2 (yellow)
in 1JFF.

7106
L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
Figure 5. MD simulation of paclitaxel (REDOR-Taxol-1JFF) (a), 1 (b) and 2 (c) in 1JFF (50 ps).
As Figure 5 shows, the critical C20–OHꢁ ꢁ ꢁN(His229) H-bond is
uncorrected. Specific optical rotations were measured on a Per-
kin–Elmer Model 241 polarimeter. TLC was performed on Merck
DC-alufolien with Kieselgel 60F-254 and column chromatography
was carried out on silica gel 60 (Merck; 230–400 mesh ASTM). Pur-
ity was determined with a Waters HPLC assembly consisting of
dual Waters 515 HPLC pumps, a PC workstation running Millen-
nium 32, and a Waters 996 PDA detector, using a Phenomenex
Curosil-B column, employing CH3CN/water (2/3) as the solvent
system with a flow rate of 1 mL/min. High-resolution mass spectra
were obtained at the Mass Spectrometry Laboratory, University of
Illinois at Urbana-Champaign, Urbana, IL.
stable in all three cases (average distance: 2.1 0.4 Å for 1;
2.3 0.7 Å for 2; 2.0 0.2 Å for REDOR-Taxol-1JFF). However, the
conformation of 1 is very flexible, that is, unstable (Fig. 5b), and
substantially deviates from that of REDOR-Taxol (Fig. 5a), espe-
cially the phenyl moiety as well as the acetoxy group, which is sup-
posed to mimic the same group at the C4 position of paclitaxel. As
Figure 5c indicates, 2 mimics REDOR-Taxol much better than 1, but
the 30-phenyl and 30N-benzoyl groups, especially the latter, move
around in a wide range, showing conformational instability. The
MD simulation analysis may indicate that the conformational
instability of the paclitaxel mimics needs to be addressed to im-
prove their potency, besides structural over-simplification.
6.1.2. Materials
In summary, novel paclitaxel-mimicking alkaloids were synthe-
sized based on the computational design, mimicking the REDOR-
Taxol structure. The alkaloid 2 bearing a 5-7-6 tricyclic scaffold
mimics REDOR-Taxol best among the compounds designed and
was found to be the most potent compound against several drug-
sensitive and drug-resistant human cancer cell lines. An MD
simulation study on the paclitaxel mimics 1 and 2 as well as
REDOR-Taxol in the 1JFF tubulin was quite informative to evaluate
the level of mimicking, as compared to static energy minimized
structures of these mimics. For example, the MD simulation study
clearly distinguishes the difference between the 5-6-6 and 5-7-6
tricyclic scaffolds, and also substantial difference in the conforma-
tional stability of the tubulin-bound structures between 2 and
REDOR-Taxol. The latter may account for the large difference in
potency, and provides critical information for possible improve-
ment in the future design of paclitaxel mimics. The comparison
of potency with the previously reported paclitaxel mimics has dis-
closed a considerable enhancement in potency by introducing non-
aromatic isoserine side chain, especially against MDR cancer cell
lines. Thus, it is anticipated that the paclitaxel mimics, combining
5-7-6 tricyclic scaffolds and a non-aromatic isoserine side chains,
would be considerably more potent than the mimics 2 and A,
The chemicals were purchased from Aldrich–Sigma Co. and
used as received or purified before use by standard methods. Tet-
rahydrofuran (THF) was freshly distilled from sodium metal and
benzophenone. Dichloromethane was distilled immediately prior
to use under nitrogen from calcium hydride. N,N-Dimethylform-
amide (DMF) was distilled over 4A molecular sieves under reduced
pressure. 4-(N,N-Dimethylamino)pyridine (DMAP) was uses as re-
ceived. The preparation of 8a–d is described in the Supplementary
data.
6.1.3. (3S,5R)-1-(tert-Butoxycarbonyl)-5-[hydroxy(2-ethenyl-6-
hydroxymethylphenyl)methyl]-3-triisopropylsiloxy-pyrrolidine
(9a)
Aryl bromide 8a (94 mg, 0.44 mmol) was desolved in dry THF
(4 mL) under N2 and 2.5 M n-BuLi in hexane (0.39 mL, 0.97 mmol)
was added at ꢀ78 °C. The resulting solution was stirred for 1 h.
Then, HMPA (0.09 mL) was added and the solution was stirred for
1 h. To this solution, a solution of aldehyde 6 (127 mg, 0.34 mmol)
in dry THF (2.8 mL) was added dropwise. The resulting mixture was
allowed to slowly warm up to room temperature with stirring over-
night. Then, the reaction was quenched with a saturated NH4Cl
solution (20 mL) and the reaction mixture was extracted with
CH2Cl2 (3 ꢂ 20 mL). The combined organic layers were washed with
brine and dried over MgSO4. The solvent was removed under re-
duced pressure, and the residue was purified by flash chromatogra-
phy on silica gel (hexanes/EtOAc = 8:1–4:1) as eluent to give 9a
(139 mg, 81% yield) as white solid: mp 38–40 °C; 1H NMR
(400 MHz, CDCl3) d 0.96 (m, 21H), 1.32–1.44 (m, 9H), 1.74 (m,
1H), 2.19 (m, 1H), 3.34 (m, 1H), 3.67 (m, 1H), 4.31 (m, 1H), 4.58
(m, 1H), 4.76 (m, 1H), 4.96 (m, 1H), 5.10 (m, 1H), 5.42 (m, 2H),
7.03 (m, 1H), 7.28 (m, 3H); 13C NMR (100.5 MHz, CDCl3) d 12.8,
18.5, 28.9, 39.4, 56.5, 57.1, 65.5, 70.9, 80.2, 81.3, 116.5, 117.7,
127.4, 127.7, 131.2, 136.5, 136.8, 140.2, 158.7. HRMS calcd. for
exhibiting IC50 values less than 1 lM against several cancer cell
lines examined in this study. Further studies along this line are ac-
tively in progress in these laboratories.
6. Experimental
6.1. Chemistry
6.1.1. General methods
1H and 13C NMR spectra were measured on a Varian 300, 400,
500 or 600 MHz NMR spectrometer. Melting points were measured
on a Thomas Hoover Capillary melting point apparatus and are
C
28H47NO5SiNa+ 528.3121, found 528.3116 (
D
= ꢀ1.0 ppm).

L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
7107
6.1.4. 1-(tert-Butoxycarbonyl)-2-[(2,6-diethenylphenyl)-
6.1.8. (3S,5R)-5-[Acetoxy(2-allyl-6-methoxymethoxymethyl-
hydroxymethyl]-4-triisopropylsiloxypyrrolidine (9c)
phenyl)methyl]-1-(tert-butoxycarbonyl)-3-triisopropylsiloxy-
pyrrolidine (10d)
A mixture of 8c (257 mg, 1.22 mmol) and Mg turnings (35 mg,
1.47 mmol) in THF (12 mL) was added 1 drop of 1,2-dibromoeth-
ane and the mixture was heated at reflux for 2 h. Then, the result-
ing Grignard reagent solution was cooled to room temperature.
Aldehyde 6 (226 mg, 0.61 mmol) was dissolved in THF (7 mL)
and cooled down to ꢀ78 °C. The Grignard reagent generated above
(1.22 mmol, 2.0 equiv) was added to the aldehyde solution by can-
nula and the reaction mixture was stirred overnight. The reaction
was quenched with saturated aqueous NH4Cl solution (20 mL)
and extracted with CH2Cl2 (20 mL ꢂ 3). The organic layer was dried
over anhydrous MgSO4 and solvent was removed under reduced
pressure. The residue was purified by column chromatography
on silica gel using hexanes/EtOAc (17:1) as the eluent to afford
9c (227 mg, 74% yield) as colorless oil: 1H NMR (400 MHz, CDCl3)
d 0.96 (m, 21H), 1.32–1.44 (m, 9H), 1.74 (m, 1H), 2.19 (m, 1H),
3.31 (m, 1H), 3.57 (m, 1H), 3.85 (m, 1H), 4.51 (m, 1H), 4.98 (m,
1H), 5.12 (m, 2H), 5.41 (m, 2H), 6.96–7.74 (m, 5H); 13C NMR
(100.5 MHz, CDCl3) d 12.0, 18.0, 28.2, 36.8, 39.6, 55.6, 63.8, 70.0,
74.1, 81.0, 114.9, 127.4, 127.9, 128.1, 136.5, 136.4, 137.9, 158.8.
Colorless oil; 70% yield for 2 steps; 1H NMR (400 MHz, CDCl3) d
1.03 (m, 21H), 1.28–1.43 (m, 9H), 1.66 (m, 1H), 2.05 (m, 4H), 3.42
(s, 3H), 3.65 (m, 4H), 4.70 (m, 6H), 5.05 (m, 2H), 5.97 (m, 1H), 6.10
(m, 1H), 7.11–7.31 (m, 3H); 13C NMR (100.5 MHz, CDCl3) d 11.9,
17.8, 21.0, 27.7, 37.6, 38.4, 55.2, 58.6, 66.9, 70.7, 72.5, 73.4, 79.7,
95.4, 115.9, 127.7, 128.1, 130.3, 133.5, 134.1, 137.1, 139.2, 154.5,
169.6. 1HRMS calcd. for C33H55NO7SiH+ 606.3826, found 606.3832
(D = 1.0 ppm).
6.1.9. (2S,10S/R,10aR)-10-Acetoxy-9-acetoxymethyl-2-triisopro-
pylsiloxy-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-5-
one (11a)
Nitrogen gas was bubbled into a solution of 10a (167 mg,
0.28 mmol) in CH2Cl2 (15 mL) at ꢀ78 °C for 3 min. Then, O3 gas
was bubbled into the solution till the color of the solution turned
blue (8 min), and N2 was bubbled into the solution for another
3 min until the blue color disappeared. Dimethyl sulfide (0.1 mL,
1.4 mmol) was added to the solution and the reaction mixture
was allowed to warm to room temperature. The reaction mixture
was stirred at room temperature for 3 h. The solvents were re-
moved in vacuo and the resulting crude aldehyde was used in
the next step without further purification.
Sodium chlorite (200 mg, 2.24 mmol) was added to a solution of
the aldehyde obtained (ꢃ0.28 mmol) and sodium phosphate
(monobasic) (350 mg, 2.24 mmol) in acetone/water (1:1, 2.4 mL).
The reaction mixture was stirred at room temperature for 1 h
and quenched with ethyl acetate (60 mL). The organic layer was
washed with hydrochloric acid (1N, 10 mL), Na2S2O3 (10%,
10 mL ꢂ 2), brine (10 mL), dried over MgSO4, filtered, and concen-
trated. The resulting crude acid was used in the next step without
further purification.
A solution of potassium hydroxide (4 g) in water (8 mL) and
ethanol (32 mL) was added to a solution of diazaldÒ (4 g) in ether
(60 mL) at 0 °C. The mixture was stirred for 10 min at 0 °C and dis-
tilled to afford a yellow ether solution. The diazomethane solution
in ether was added to the solution of the acid obtained
(ꢃ0.28 mmol) in ether (12 mL) until the yellow color did not disap-
pear. The mixture was stirred for 10 min and the reaction was
quenched with acetic acid. The solvents were removed in vacuo
and the resulting crude methyl ester was used in the next step
without further purification.
HRMS calcd. for
= 4.0 ppm).
C
29H46NO4SiH+ 502.3353, found 502.3373
(D
6.1.5. (3S,5R)-5-[Acetoxy(2-acetoxymethyl-6-ethenyl-phenyl)
methyl]-1-(tert-butoxycarbonyl)-3-triisopropylsiloxypyrroli-
dine (10a)
To a solution of 9a (134 mg, 0.26 mmol) and DMAP (4.8 mg,
0.06 mmol) in CH2Cl2 (2.0 mL) was added triethylamine (0.41 mL,
1.4 mmol) and acetic anhydride (0.27 mL, 0.84 mmol) at 0 °C. The
reaction mixture was stirred overnight, quenched with saturated
aqueous NH4Cl solution (20 mL) and extracted with CH2Cl2
(30 mL ꢂ 3). The organic layer was dried over anhydrous MgSO4
and the solvent was removed under reduced pressure to afford a
liquid residue. The residue was purified by silica gel column chro-
matography on silica gel using hexanes/EtOAc (8:1) as the eluent
to afford 10a (150 mg, 97% yield) as colorless oil: 1H NMR
(400 MHz, CDCl3) d 1.04 (m, 21H), 1.28–1.43 (m, 9H), 1.70 (m,
1H), 2.06–2.10 (m, 7H), 3.40 (m, 1H), 3.62 (m,1H), 4.57 (m, 1H),
4.79 (m, 1H), 5.31 (m, 3H), 5.54 (m, 2H), 6.03 (m, 1H), 7.17–7.52
(m, 4H); 13C NMR (100.5 MHz, CDCl3) d 12.1, 17.9, 20.9, 28.0,
28.4, 38.3, 55.0, 58.5, 64.3, 71.0, 73.9, 79.5, 116.6, 128.2, 128.7,
129.9, 132.3, 134.2, 136.5, 139.4, 154.6, 170.3, 170.7. HRMS calcd.
for C32H51NO7SiNa+ 612.3333, found 612.3329 (
D
= ꢀ0.6 ppm).
To a solution of crude ester obtained (ꢃ0.28 mmol) in CH2Cl2
(1.5 mL) at 0 °C was added trifluoroacetic acid (1.5 mL). The mixture
was stirred at 0 °C for 30 min and room temperature for 30 min. The
solvent and the acid were removed under reduced pressure and the
residue was used in next step without further purification.
To a solution of the residue in ethyl acetate (14 mL) was added
saturated aqueous sodium bicarbonate (14 mL). The mixture was
stirred vigorously overnight and the reaction was quenched with
ethyl acetate (50 mL). The reaction mixture was washed with
water (10 mL) saturated aqueous ammonium chloride (10 mL),
water (10 mL) and brine (10 mL). The organic layer was dried over
anhydrous MgSO4 and the solvent was removed under reduced
pressure. The residue was purified by column chromatography
on silica gel using hexanes/EtOAc (3:1–2:1) as the eluent to afford
6.1.6. (3S,5R)-2-[Acetoxy(2-methoxymethoxymethyl-6-ethenyl-
phenyl)methyl]-1-(tert-butoxycarbonyl)-3-triisopropylsiloxy-
pyrrolidine (10b)
Colorless oil; 70% yield for 2 steps; 1H NMR (400 MHz, CDCl3) d
1.02 (m, 21H), 1.28–1.43 (m, 9H), 2.06–2.10 (m, 5H), 3.40 (m, 4H),
3.59 (m, 1H), 4.57 (m, 2H), 4.79 (m, 4H), 5.28 (d, J = 10.8 Hz, 1H),
5.51 (d, J = 17.6, 1H), 6.02–6.22 (m, 1H), 7.17–7.52 (m, 4H); 13C
NMR (100.5 MHz, CDCl3) d 12.1, 17.9, 20.9, 28.0, 28.4, 38.3, 54.7,
55.0, 58.6, 66.6, 70.2, 72.7, 79.7, 95.4, 116.0, 127.2, 127.5, 128.9,
129.1, 133.5, 136.4, 136.8, 137.0, 138.6, 154.4, 169.5. HRMS calcd.
for C32H53NO7SiH+ 592.3670, found 592.3663 (
D
= ꢀ1.2 ppm)
6.1.7. (3S,5R)-5-[Acetoxy(2,6-diethenylphenyl)methyl]-1-(tert-
butoxycarbonyl)-3-triisopropylsiloxypyrrolidine (10c)
11a-a and 11a-b (1:2) in 50% yield for 5 steps.
Colorless oil; 98% yield; 1H NMR (400 MHz, CDCl3) d 1.09 (m,
21H), 1.28–1.47 (m, 9H), 1.66 (m, 1H), 1.90–2.10 (m, 4H), 3.48
(m, 2H), 4.43 (m, 1H), 4.79 (m, 1H), 5.23 (m, 2H), 5.47 (m, 2H),
6.43 (m, 1H), 7.27–7.74 (m, 5H). 13C NMR (100.5 MHz, CDCl3) d
12.7, 18.5, 21.1, 28.7, 37.5, 55.5, 59.4, 71.5, 73.9, 79.4, 116.6,
128.2, 128.7, 129.5, 132.8, 137.4, 139.4, 155.8, 169.4. HRMS calcd.
6.1.9.1. (2S,10S,10aR)-10-Acetoxy-9-acetoxymethyl-2-triisopro-
pylsiloxy-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-5-
one (11a-
a
). Colorless oil; 17% for 5 steps; ½a D22
ꢀ80.4 (c 1.1,
ꢄ
CHCl3); 1H NMR (400 MHz, CDCl3) d 1.09 (m, 21H), 2.05 (s, 3H),
2.06 (m, 2H), 2.21 (s, 3H), 3.80 (d, J = 3.2 Hz, 2H), 4.15 (td,
J = 10.8, 5.6 Hz, 1H), 4.61 (s, 1H), 5.09 (d, J = 12.8 Hz, 1H), 5.33 (d,
for C31H48NO5SiH+ 544.3458, found 544.3472 (
D = 2.6 ppm).

7108
L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
J = 12.8 Hz, 1H), 6.38 (d, J = 12.8, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.50
(d, J = 1.2 Hz, 1H), 8.15 (dd, J = 7.6, 1.6 Hz, 1H); 13C NMR
(100.5 MHz, CDCl3) d 12.2, 18.2, 20.8, 21.1, 42.2, 55.3, 58.9, 65.8,
69.5, 73.1, 128.6, 129.2, 130.8, 132.9, 134.2, 135.5, 162.3, 170.4,
171.1. HRMS calcd. for C26H39NO6SiH+ 490.2625, found 490.2621
1.05 (m, 21H), 2.02 (dt, J = 13.6, 2.8 Hz, 1H), 2.06 (s, 3H), 2.26
(dd, J = 10.0, 4.0 Hz, 1H), 3.38 (s, 3H), 3.38 (d, J = 11.2 Hz, 1H),
3.47 (dd, J = 10.4, 3.6 Hz, 1H), 3.52 (d, J = 10.0 Hz, 1H), 4.36 (m,
2H), 4.46 (d, J = 10.0 Hz, 1H), 4.52 (d, J = 9.6 Hz, 1H), 4.65 (dd,
J = 10.8, 5.2 Hz, 2H), 4.84 (d, J = 9.6 Hz, 1H), 6.41 (d, J = 2.4 Hz,
1H), 7.26 (m, 3H); 13C NMR (100.5 MHz, CDCl3) d 11.9, 17.8, 21.1,
41.9, 42.7, 55.4, 55.9, 61.6, 67.3, 67.9 70.9, 95.6, 129.2, 129.3,
129.9, 131.9, 137.5, 137.6, 169.9, 170.6. HRMS calcd. for
(D
= ꢀ0.8 ppm).
6.1.9.2. (2S,10R,10aR)-10-Acetoxy-9-acetoxymethyl-2-triisopro-
pylsiloxy-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-5-
C
27H43NO6SiH+ 506.2938, found 506.2928 (
D
= ꢀ2.0 ppm).
one (11a-b). Colorlessoil;33%yieldforfivesteps;½a D22
ꢄ ꢀ137(c0.9,
CHCl3); 1H NMR (400 MHz, CDCl3) d 1.09 (m, 21H), 1.83 (td, J = 12.4,
4.0 Hz, 1H), 2.02 (s, 3H), 2.07 (s, 3H), 2.13 (ddd, J = 18.4, 7.2, 4.8 Hz,
1H), 3.72 (d, J = 13.2 Hz, 1H), 3.80 (dd, J = 12.8, 4.4, 1H), 4.31 (ddd,
J = 10.8, 5.6, 2.4 Hz, 1H), 4.66 (t, J = 3.6 Hz, 1H), 5.17 (d, J = 12.4 Hz,
1H), 5.28 (d, J = 12.8 Hz, 1H), 6.40 (d, J = 2.4 Hz, 1H), 7.55 (m, 2H),
8.18 (dd, J = 7.2, 1.6 Hz, 1H); 13C NMR (100.5 MHz, CDCl3) d 12.0,
17.9, 20.8, 20.9, 37.8, 54.6, 57.5, 63.1, 63.7, 69.5, 128.5, 129.8,
131.5, 133.5, 133.8, 162.0, 170.0, 170.4. HRMS calcd. for C26H39NO6-
6.1.12. (2S,10S,10aR)-10-Hydroxy-9-hydroxymethyl-2-triisopro-
pylsiloxy-2,3,10,10a-tetrahydro-1H-pyrrolo-[1,2-b]isoquino-
lin-5-one (12)
To a solution of 11a-a (133 mg, 0.27 mmol) in methanol
(10.9 mL) and water (5.5 mL) was added potassium carbonate
(115 mg, 0.70 mmol). The mixture was stirred at room tempera-
ture for 35 min. Then, the reaction mixture was quenched with
ethyl acetate (50 mL) and the organic layer was washed with satu-
rated aqueous NH4Cl solution (10 mL), water (10 mL ꢂ 2) and brine
(10 mL). The organic layer was dried over anhydrous MgSO4 and
solvent was removed under reduced pressure. The residue was
purified by column chromatography on silica gel using 3% metha-
nol in chloroform as the eluent to afford 12 as a white solid (92 mg,
SiH+ 490.2625, found 490.2618 (
D
= ꢀ1.4 ppm).
6.1.10. (2S,10S/R,10aR)-10-Acetoxy-9-methoxymethoxy-methyl-
2-triisopropylsiloxy-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]-
isoquinolin-5-one (11b)
Colorless oil (
a
/b = 3:4 by 1H NMR); 50% yield for 5 steps; 11b
a:
84% yield): mp 153–155 °C; ½a D22
ꢄ
ꢀ86 (c 1.5, CHCl3); 1H NMR
1H NMR (400 MHz, CDCl3) d 1.06 (m, 21H), 2.11 (m, 2H), 2.21 (s,
3H), 3.31 (s, 3H), 3.79 (m, 2H), 4.14 (td, J = 10.8, 5.6 Hz, 1H), 4.61
(m, 6H), 4.80 (d, J = 12.8 Hz, 1H), 6.32 (d, J = 10.4, 1H), 7.40 (t,
J = 7.6 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H), 8.10 (dd, J = 8.0, 1.6 Hz,
1H). HRMS calcd. for C26H41NO6SiH+ 492.2781, found 492.2775
(400 MHz, CDCl3) d 1.06 (m, 21H), 1.85 (ddd, J = 14.8, 10.8,
4.0 Hz, 1H), 2.48 (dd, J = 12.8, 5.6 Hz, 1H), 3.68 (d, J = 13.6 Hz,
1H), 3.72 (dd, J = 13.2, 4.4 Hz, 1H), 4.02 (td, J = 16.4, 5.2 Hz, 1H),
4.61 (t, J = 3.6 Hz, 1H), 4.71 (t, J = 12.4 Hz, 1H), 4.83 (m, 2H), 5.79
(br s, 1H), 7.22 (m, 2H), 7.78 (dd, J = 7.6, 3.6 Hz, 1H); 13C NMR
(100.5 MHz, CDCl3) d 11.9, 17.9, 42.3, 55.3, 59.8, 65.1, 69.4, 73.3
127.5, 127.8, 129.5, 133.8, 137.3, 139.7, 162.9. HRMS: m/e calcd
(D
= ꢀ1.2 ppm).
6.1.11. (2S,10S/R,10aR)-10-Acetoxy-9-methoxycarbonyl-2-triiso-
propylsiloxy-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquin-
olin-5-one (11c)
for C22H35NO4SiH+: 406.2414, found: 406.2411 (
D
= ꢀ0.6 ppm).
6.1.13. (2S,10S,10aR)-9-(tert-Butyldimethylsiloxymethyl)-10-
hydroxy-2-triisopropylsiloxy-2,3,10,10a-tetrahydro-1H-
pyrrolo[1,2-b]isoquinolin-5-one (13)
Colorless oil (a
/b = 3:2 by 1H NMR); 63% yield for 5 steps; 1H
NMR (400 MHz, CDCl3) d 1.04 (m, 21H), 1.89 (td, J = 12.8, 4.0,
0.5H), 1.98 (s, 1.5H), 2.16 (m, 2H), 3.77 (m, 2H), 3.84 (s, 1.5H),
3.92 (s, 1.5H), 4.07 (m, 0.5H), 4.30 (ddd, J = 11.2, 5.6, 2.4 Hz,
0.5H), 4.64 (m, 1H), 6.40 (d, J = 10.8, 0.5H), 6.74 (d, J = 2.4 Hz,
0.5H), 7.49 (t, J = 8.4 Hz, 0.5H), 7.58 (t, J = 7.6 Hz, 0.5H), 7.77 (dd,
J = 8.0, 1.6 Hz, 0.5H), 8.04 (dd, J = 8.0, 1.6 Hz, 0.5 Hz), 8.24 (dd,
J = 8.0, 1.6 Hz, 0.5H), 8.36 (dd, J = 7.6, 1.2 Hz, 0.5H); 13C NMR
(100.5 MHz, CDCl3) d 12.3, 12.5, 17.9, 20.6, 20.8, 38.2, 42.2, 52.4,
52.6, 54.7, 54.9, 57.6, 58.9, 64.6, 69.2, 69.5, 71.8, 128.3, 129.4,
130.1, 130.2, 130.3, 130.8, 131.7, 131.9, 132.9, 133.5, 134.9,
136.7, 161.5, 161.7, 166.9, 168.6, 169.7, 170.1. HRMS calcd. for
To a solution of 12 (90 mg, 0.22 mmol) and imidazole (60 mg,
0.88 mmol) in dry DMF (0.6 mL) was added t-butyldimethylsilyl
chloride (0.29 mmol in 1.2 mL DMF) dropwise via syringe at 0 °C.
The reaction mixture was stirred for 35 min at room temperature
and quenched with saturated aqueous NH4Cl solution (10 mL).
The reaction mixture was extracted with EtOAc (20 mL ꢂ 3),
washed with water (10 mL ꢂ 2), and brine (10 mL), dried over
MgSO4 and concentrated in vacuo. The crude product was purified
on a silica gel column using hexanes/EtOAc (2:1) as the eluent to
give 13 as a white solid (113 mg, 99% yield): mp 183–184 °C;
C
26H41NO6SiH+ 492.2781, found 492.2775 (
D
= ꢀ1.2 ppm).
½
a 2D2
ꢄ
ꢀ110 (c 2.5, CHCl3); 1H NMR (400 MHz, CDCl3) d ꢀ0.01 (s,
3H), 0.09 (s, 3H), 0.87 (s, 9H), 1.04 (m, 21H), 1.93 (td, J = 12.8,
4.0 Hz, 1H), 2.51 (dd, J = 12.8, 5.6 Hz, 1H), 3.72 (d, J = 12.8 Hz,
1H), 3.84 (dd, J = 12.8, 4.4 Hz, 1H), 4.09 (td, J = 10.8, 5.6 Hz, 1H),
4.64 (s, 1H), 4.74 (d, J = 12.8 Hz, 1H), 4.88 (dd, J = 10.8, 3.6, 1H),
5.18 (d, J = 12.8 Hz, 1H), 5.61 (d, J = 4.4 Hz, 1H), 7.28 (m, 2H),
8.04 (dd, J = 7.2, 2.0 Hz, 1H); 13C NMR (100.5 MHz, CDCl3) d ꢀ5.4,
ꢀ5.1, 12.0, 17.9, 18.1, 25.6, 42.5, 55.2, 59.6, 66.2, 69.6, 73.8,
127.3, 128.3, 130.4, 132.7, 136.1, 139.9, 162.5. HRMS: m/e calcd
6.1.11.1.
thyl)-2-triisopropylsiloxy-2,3,11,11a-tetra-hydro-1H-benzo[d]-
(2S,11S,11aR)-11-Acetoxy-10-(methoxymethoxyme-
pyrrolo[1,2-a]azepin-5(6H)-one (11d-a). Colorless oil; 63% yield
for 5 steps; ½a 2D2
ꢄ
ꢀ26 (c 1.8, CHCl3); 1H NMR (400 MHz, CDCl3) d
1.01 (m, 21H), 1.99 (m, 2H), 2.07 (s, 3H), 3.36 (s, 3H), 3.46 (d,
J = 13.2 Hz, 1H), 3.75 (d, J = 17.2 Hz, 1H), 3.98 (dd, J = 12.8, 5.6 Hz,
1H), 4.18 (dd, J = 10.8, 6.0 Hz, 1H), 4.38 (d, J = 17.2 Hz, 1H), 4.45
(t, J = 3.6 Hz, 1H), 4.50 (d, J = 11.6 Hz, 1H), 4.61 (dd, J = 12.8,
6.4 Hz, 2H), 5.05 (d, J = 11.6 Hz, 1H), 6.43 (s, 1H), 7.14 (dd, J = 6.0,
2.4 Hz, 1H), 7.25 (m, 2H); 13C NMR (100.5 MHz, CDCl3) d 11.9,
17.9, 20.9, 40.9, 43.8, 55.4, 57.9, 59.8, 67.5, 68.9, 95.3, 128.9,
129.4, 131.2, 134.6, 135.6, 135.7, 167.5, 170.2. HRMS calcd. for
for C28H49NO4Si2H+: 520.3278, found: 520.3279 (
D = 0.1 ppm).
6.1.14. (2S,10S,10aR)-10-Benzoyloxy-9-(tert-butyldimethyl-
siloxymethyl)-2-triisopropylsiloxy-1,2,3,5,10,10a-hexahydro-
pyrrolo[1,2-b]isoquinolin-5-one (14)
To a solution of 13 (60 mg, 0.115 mmol) and DMAP (4.8 mg,
0.06 mmol) in CH2Cl2 (1.0 mL) was added triethylamine (0.13 mL,
0.92 mmol) and benzoyl chloride (0.1 mL, 0.69 mmol) at 0 °C. The
reaction mixture was stirred overnight, quenched with saturated
aqueous NH4Cl solution (20 mL) and extracted with CH2Cl2
(30 mL ꢂ 3). The organic layer was dried over anhydrous MgSO4
C
27H43NO6SiH+ 506.2938, found 506.2928 (
D
= ꢀ2.0 ppm).
6.1.11.2.
(2S,11R,11aR)-11-Acetoxy-10-(methoxymethoxyme-
thyl)-2-triisopropylsiloxy-2,3,11,11a-tetrahydro-1H-benzo[d]-
pyrrolo[1,2-a]azepin-5(6H)-one (11d-b). Colorless oil; 6% yield
for 5 steps; ½a 2D2
ꢄ
+62 (c 0.2, CHCl3); 1H NMR (400 MHz, CDCl3) d

L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
7109
and solvent was removed under reduced pressure to afford a liquid
residue. The residue was purified by column chromatography on
silica gel using hexanes/EtOAc (6:1) as the eluent to afford 14
3) and brine (3 mL). The organic layer was dried over anhydrous
MgSO4 and solvent was removed under reduced pressure. The resi-
due was purified by column chromatography using hexanes/EtOAc
(1:4) as the eluent to afford 17a as white solid (23 mg, 100% yield):
(73 mg, 100%) as colorless oil: ½a D22
ꢄ
ꢀ110 (c 1.3, CHCl3); 1H NMR
(400 MHz, CDCl3) d ꢀ0.22 (s, 3H), 0.21 (s, 3H), 0.75 (s, 9H), 1.02
(m, 21H), 2.15 (m, 2H), 3.85 (m, 2H), 4.30 (td, J = 10.8, 6.0 Hz,
1H), 4.63 (s, 1H), 4.67 (d, J = 14.4 Hz, 1H), 4.76 (d, J = 14.0, 1H),
6.60 (d, J = 10.8 Hz, 1H), 7.48 (m, 3H), 7.64 (m, 2H), 8.13 (m, 3H);
13C NMR (100.5 MHz, CDCl3) d ꢀ5.7, ꢀ5.8, 12.0, 17.9, 18.2, 25.7,
41.9, 55.1, 58.7, 63.2, 69.3, 73.2, 127.2, 128.3, 128.7, 129.2, 129.7,
129.8, 130.7, 133.7, 133.8, 138.9, 162.6, 165.5. HRMS: m/e calcd
mp 76–77 °C; ½a 2D2
ꢄ
ꢀ239 (c 10.3, CHCl3); 1H NMR (400 MHz, CDCl3) d
1.90 (s, 3H), 2.24 (m, 2H), 3.83 (dd, J = 11.2, 3.2 Hz, 2H), 4.32 (td,
J = 8.4, 4.4 Hz, 1H), 4.64 (t, J = 3.2 Hz, 1H), 5.04 (d, J = 10.0, 1H),
5.21 (d, J = 10.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 7.48 (m, 4H), 7.64
(t, J = 6.0 Hz, 1H), 8.10 (d, J = 6.0 Hz, 2H), 8.18 (dd, J = 6.0, 1.2 Hz,
1H); 13C NMR (100.5 MHz, CDCl3) d 20.6, 41.2, 54.8, 58.9, 65.6,
68.8, 73.4, 128.8, 129.0, 129.1, 129.2, 130.0, 130.7, 133.4, 134.1,
134.6, 135.9, 162.6, 165.9, 170.6. HRMS: m/e calcd for C22H21NO6H+:
for C35H53NO5Si2H+: 624.3541, found: 624.3516 (
D
= ꢀ3.9 ppm).
396.1447, found: 396.1436 (
D
= ꢀ2.8 ppm).
6.1.15. (2S,10S,10aR)-10-Benzoyloxy-2-triisopropylsiloxy-
1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-5-one (15)
To a solution of 14 (134 mg, 0.21 mmol) in ethanol (10 mL) was
added 0.5 N HCl in ethanol (2 mL). The mixture was stirred at room
temperature for 2.5 h. Then, the reaction mixture was quenched
with saturated aqueous NaHCO3 solution (20 mL) and extracted
with CH2Cl2 (30 mL ꢂ 3). The organic layer was washed with water
(10 mL) and brine (10 mL). The organic layer was dried over anhy-
drous Na2SO4 and solvent was removed under reduced pressure.
The residue was purified by column chromatography on silica gel
using hexanes/EtOAc (2:1) as the eluent to afford 15 as a white so-
6.1.18. (2S,10S,10aR)-10-Benzoyloxy-9-pent-4-enoyloxymethyl-
2-triisopropylsiloxy-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]iso-
quinolin-5-one (16b)
Colorless oil; 100% yield; ½a D22
ꢄ
ꢀ158 (c 2.0, CHCl3); 1H NMR
(400 MHz, CDCl3) d 1.00 (m, 21H), 2.22 (m, 6H), 3.83 (m, 2H),
4.32 (dd, J = 18.0, 8.8 Hz, 1H), 4.63 (d, J = 2.0 Hz, 1H), 4.92 (dd,
J = 10.4, 1.6 Hz, 1H), 4.98 (t, J = 5.6 Hz, 1H), 5.06 (d, J = 12.8, 1H),
5.22 (d, J = 12.8 Hz, 1H), 5.72 (m, 1H), 6.68 (d, J = 10.8 Hz, 1H),
7.49 (m, 4H), 7.64 (t, J = 7.6 Hz, 1H), 8.10 (dd, J = 8.4, 1.2 Hz, 2H),
8.20 (dd, J = 7.2, 1.6 Hz, 1H); 13C NMR (100.5 MHz, CDCl3) d 11.9,
17.9, 28.5, 32.9, 41.9, 55.1, 58.9, 65.1, 69.3, 73.2, 115.4, 128.4,
128.7, 128.9, 129.0, 129.7, 130.6, 133.2, 133.8, 134.3, 135.7,
136.5, 162.2, 165.7, 172.3. HRMS: m/e calcd for C34H45NO6SiH+:
lid (102 mg, 95% yield): mp 95–98 °C; ½a D22
ꢄ
ꢀ130 (c 1.3, CHCl3); 1H
NMR (400 MHz, CDCl3) d 0.98 (m, 21H), 2.17 (m, 2H), 2.68 (br s,
1H), 3.75 (d, J = 12.8 Hz, 1H), 3.88 (dd, J = 12.8, 4.4 Hz, 1H), 4.30
(td, J = 10.8, 6.0 Hz, 1H), 4.55 (d, J = 13.6 Hz, 1H), 4.62 (d, J = 13.6,
1H), 4.66 (s, 1H), 6.53 (d, J = 10.8 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H),
7.49 (t, J = 8.0 Hz, 2H), 7.61 (m, 2H), 7.86 (dd, J = 7.6, 1.2 Hz, 1H),
8.10 (dd, J = 8.4, 1.2 Hz, 2H); 13C NMR (100.5 MHz, CDCl3) d 11.9,
17.9, 42.0, 55.1, 58.8, 63.1, 69.2, 73.0, 127.5, 128.4, 128.7, 129.1,
129.8, 132.1, 133.7, 134.1, 138.9, 162.7, 165.9. HRMS: m/e calcd
592.3094, found: 592.3077 (
D
= ꢀ2.9 ppm).
6.1.19. (2S,10S,10aR)-10-Benzoyloxy-2-hydroxy-9-pent-4-enoyl-
oxymethyl-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-
5-one (17b)
White solid; 97% yield; mp 50–52 °C; ½a D22
ꢀ227 (c 1.5, CHCl3);
ꢄ
for C29H39NO5SiH+: 510.2676, found: 510.2672 (
D
= ꢀ0.7 ppm).
1H NMR (400 MHz, CDCl3) d 2.22 (m, 6H), 3.85 (d, J = 2.4 Hz, 2H),
4.33 (td, J = 10.4, 5.6 Hz, 1H), 4.61 (t, J = 2.4 Hz, 1H), 4.92 (dd,
J = 10.4, 1.6 Hz, 1H), 4.95 (dd, J = 13.2, 1.6 Hz, 1H), 5.06 (d,
J = 12.8 Hz, 1H), 5.21 (d, J = 12.8 Hz, 1H), 5.70 (m, 1H), 6.68 (d,
J = 10.8 Hz, 1H), 7.48 (m, 4H), 7.63 (t, J = 7.3 Hz, 1H), 8.08 (d,
J = 8.4, 1.2 Hz, 2H), 8.14 (dd, J = 7.2, 1.2 Hz, 1H); 13C NMR
(100.5 MHz, CDCl3) d 28.5, 32.9, 40.9, 54.5, 58.7, 65.1, 68.4, 73.1,
115.5, 128.5, 128.7, 128.8, 128.9, 129.8, 130.4, 133.3, 133.9,
134.3, 135.7, 136.5, 162.6, 165.6, 172.3. HRMS: m/e calcd for
6.1.16. (2S,10S,10aR)-9-Acetoxymethyl-10-benzoyloxy-2-triiso-
propylsiloxy-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquin-
olin-5-one (16a)
To a solution of 15 (32 mg, 0.06 mmol) and DMAP (1 mg, 0.005
mmol) in CH2Cl2 (0.5 mL) was added triethylamine (0.08 mL, 0.24
mmol) and acetic anhydride (0.05 mL, 0.12 mmol) at 0 °C. The
reaction mixture was stirred overnight, quenched with saturated
aqueous NH4Cl solution (20 mL) and extracted with CH2Cl2
(20 mL ꢂ 3). The organic layer was dried over anhydrous MgSO4
and solvent was removed under reduced pressure. The residue
was purified by column chromatography on silica gel using hex-
anes/EtOAc (2:1) as the eluent to afford 16a (35 mg, 100%) as a col-
C
25H25NO6H+: 436.1760, found: 436.1750 (
D
= ꢀ2.3 ppm).
6.1.20. (2S,11R,11aR)-11-Hydroxy-10-(methoxymethoxy-
methyl)-2-triisopropylsiloxy-2,3,11,11a-tetrahydro-1H-
benzo[d]pyrrolo[1,2-a]azepin-5(6H)-one (18)
orless oil: ½a 2D2
ꢄ
ꢀ164 (c 14.1, CHCl3); 1H NMR (400 MHz, CDCl3) d
To a solution of 11d (260 mg, 0.51 mmol) in methanol (20 mL)
and water (10 mL) was added potassium carbonate (180 mg,
1.02 mmol). The mixture was stirred at room temperature for
30 min. Then, the reaction mixture was quenched with ethyl ace-
tate and the organic layer was washed with saturated aqueous
NH4Cl solution (20 mL), water (10 mL ꢂ 2) and brine (10 mL). The
organic layer was dried over anhydrous MgSO4 and solvent was re-
moved under reduced pressure. The residue was purified by col-
umn chromatography on silica gel using hexanes/EtOAc (3:1) as
the eluent to afford 18 as a white solid (189 mg, 81% yield): mp
104–105 °C; 1H NMR (400 MHz, CDCl3) d 1.05 (m, 21H), 2.20 (m,
1H), 2.54 (m, 1H), 3.35 (d, J = 14.0 Hz, 1H), 3.40 (s, 3H), 3.42 (d,
J = 5.2 Hz, 1H), 3.64 (dd, J = 11.6, 3.6 Hz, 1H), 4.36 (d, J = 14.0 Hz,
1H), 4.42 (dd, J = 15.6, 6.4 Hz, 1H), 4.52 (d, J = 4.4 Hz, 1H), 4.56 (d,
J = 11.2 Hz, 1H), 4.71 (dd, J = 11.6, 6.4 Hz, 2H), 4.82 (d, J = 2.8 Hz,
1H), 4.85 (s, 1H), 7.26 (m, 3H); 13C NMR (100.5 MHz, CDCl3) d
12.0, 17.9, 43.0, 43.7, 53.9, 55.8, 60.5, 68.5, 69.4, 73.1, 95.6,
128.2, 130.9, 131.9, 133.7, 136.9, 138.6, 172.2. HRMS: m/e calcd
1.05 (m, 21H), 1.89 (s, 3H), 2.17 (m, 2H), 3.83 (td, J = 12.8, 3.6 Hz,
1H), 4.32 (dd, J = 16.4, 9.2 Hz, 1H), 4.63 (d, J = 2.0 Hz, 1H), 5.02 (d,
J = 12.8, 1H), 5.20 (d, J = 12.4 Hz, 1H), 6.70 (d, J = 10.8 Hz, 1H),
7.48 (m, 4H), 7.64 (ddd, J = 8.8, 2.4, 1.2 Hz, 1H), 8.10 (dd, J = 8.4,
1.2 Hz, 2H), 8.20 (dd, J = 6.8, 2.4 Hz, 1H); 13C NMR (100.5 MHz,
CDCl3) d 11.9, 17.9, 20.4, 41.9, 55.1, 58.8, 65.3, 69.3, 73.1, 128.5,
128.7, 129.0, 129.1, 129.7, 130.6, 133.0, 133.8, 134.3, 135.7,
162.2, 165.7, 170.3. HRMS: m/e calcd for
552.2781, found: 552.2781 (
= ꢀ0.1 ppm).
C
31H41NO6SiH+:
D
6.1.17. (2S,10S,10aR)-9-Acetoxymethyl-10-benzoyloxy-2-hydr-
oxyl-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-5-one
(17a)
To a solution of 16a (31 mg, 0.056 mmol) in CH3CN (0.6 mL) and
pyridine (0.6 mL) was added HF-pyridine (70:30, 0.31 ml) and the
mixture was stirred overnight. The reaction mixture was diluted
with EtOAc (40 mL) and washed with saturated aqueous NaHCO3
solution (10 mL ꢂ 2), CuSO4 solution (10 mL ꢂ 3), water (10 mL ꢂ
for C25H41NO5SiH+: 464.2832, found: 464.2837 (
D = 1.1 ppm).

7110
L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
6.1.21. (2S,11S,11aR)-10-(Acetoxymethyl)-11-benzoyloxy-2-tri-
isopropylsiloxy-2,3,11,11a-tetrahydro-1H-benzo[d]pyrrolo[1,2-
a]azepin-5(6H)-one (16c)
128.7, 128.8, 130.0, 132.0, 132.3, 133.2, 138.1, 164.6, 174.0,
190.9. HRMS: m/e calcd for
24H22NO5H+: 404.1498, found:
404.1495 (
= ꢀ0.7 ppm).
C
D
To a solution of 18 (125 mg, 0.27 mmol), benzoic acid (40 mg,
0.32 mmol) and triphenylphosphine (78 mg, 0.30 mmol) in THF
(1 mL) was added diisopropyl azodicarboxylate (0.060 mL, 0.30
mmol). The mixture was stirred overnight and refluxed for 3 days.
Solvent was removed under reduced pressure and the residue
was purified by column chromatography on silica gel using hex-
anes/EtOAc (5:1) to afford the corresponding Mitsunobu coupling
product, (2S,11R,11aR)-11-benzoyloxy-10-(methoxy-methoxymethyl)-
2-triisopropyl-siloxy-2,3,11,11a-tetra-hydro-1H-benzo[d]pyrrolo[1,2-
a]-azepin-5(6H)-one (47 mg, conversion 36%) accompanied by
impurity and the starting material 18 (80 mg).
To a solution of the Mitsunobu coupling product thus obtained
and anisole (0.2 mL) in CH2Cl2 (2.0 mL) was added trifluoroacetic
acid (2.0 mL) at 0 °C. The reaction mixture was stirred 2 h at room
temperature and diluted with ethyl acetate (50 mL). The organic
layer was washed by saturated NaHCO3 solution (10 mL ꢂ 2) and
NaCl (10 ml), dried over anhydrous MgSO4, and solvent was re-
moved under reduced pressure to afford a liquid residue. The res-
idue was purified by column chromatography on silica gel using
hexanes:EtOAc (2:1) as the eluent to afford 19 (32 mg, 63% yield
in 2 steps) as colorless oil.
6.1.24. (4S,5R)-4-(2-Allyloxyphenyl)-3-benzoyl-2-(4-methoxy-
phenyl)oxazolidine-5-carboxylic acid (20b)
White solid; 88% yield; mp 47–49 °C; 1H NMR (400 MHz, CDCl3)
d 3.83 (s, 3H), 4.47 (dd, J = 12.8, 3.6 Hz, 1H), 4.55 (dd, J = 13.2,
5.2 Hz, 1H), 4.91 (s, 1H), 5.18 (d, J = 10.8 Hz, 1H), 5.25 (d,
J = 17.2 Hz, 1H), 5.59 (s, 1H), 5.92 (m, 1H), 6.88 (m, 4H), 7.03 (m,
1H), 7.20 (m, 3H), 7.28 (m, 4H), 7.59 (s, 2H), 8.71 (br s, 1H); 13C
NMR (100.5 MHz, CDCl3) d 55.2, 60.5, 68.9, 81.4, 90.7, 111.6,
113.3, 117.7, 120.4, 127.0, 128.2, 128.6, 129.0, 129.2, 129.9,
130.7, 132.7, 135.5, 154.9, 159.9, 170.4. HRMS: m/e calcd for
C
27H25NO6H+: 460.1760, found: 460.1754 (
D
= ꢀ1.3 ppm).
6.1.25. (2S,10S,10aR)-9-Acetoxymethyl-10-benzoyloxy-5-oxo-1,
2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-2-yl (40S,50R)-
30-benzoyl-20-(4-methoxy-phenyl)-40-phenyloxazolidine-50-
carboxylate (21a)
To
a solution of 17a (20 mg, 0.053 mmol), 20a (21 mg,
0.053 mmol) and DMAP (3.5 mg, 0.026 mmol) in CH2Cl2 (0.5 mL)
was added EDC (22 mg, 0.11 mmol) and the reaction mixture
was stirred overnight. The mixture was then quenched with EtOAc
(50 mL) and washed with water (10 mL ꢂ 2) and brine (10 mL). The
organic layer was dried over anhydrous MgSO4, filtered, and sol-
vent was removed under reduced pressure. The residue was puri-
fied by column chromatography on silica gel using hexanes/
EtOAc (1:2) as the eluent to afford 21a as a white solid (27 mg,
82% yield based on 85% conversion): mp 109–111 °C; 1H NMR
(400 MHz, CDCl3) d 1.90 (s, 3H), 2.32 (dd, J = 14.0, 5.2 Hz, 1H),
2.42 (td, J = 10.8, 4.4 Hz, 1H), 3.80 (s, 3H), 3.89 (d, J = 14.8 Hz,
1H), 4.05 (d, J = 14.4, 4.8, 1H), 4.17 (td, J = 10.8, 5.6 Hz, 1H), 4.81
(d, J = 2.0 Hz, 1H), 5.05 (d, J = 11.6 Hz, 1H), 5.22 (d, J = 12.8 Hz,
1H), 5.36 (br s, 1H), 5.55 (t, J = 4.0 Hz, 1H), 6.71 (d, J = 10.8 Hz,
1H), 6.81 (d, J = 8.4 Hz, 2H), 7.25 (m, 11H), 7.47 (m, 4), 7.63 (t,
J = 7.6 Hz, 1H), 8.12 (d, J = 7.2 Hz, 2H), 8.21 (dd, J = 7.2, 2.0 Hz,
1H); 13C NMR (100.5 MHz, CDCl3) d 20.4, 38.3, 51.8, 55.3, 58.8,
65.4, 72.9, 73.0, 113.5, 127.0, 127.1, 128.1, 128.2, 128.6, 128.7,
128.7, 128.8, 129.0, 129.8, 129.8, 130.2, 130.7, 133.3, 134.0,
134.6, 135.2, 135.4, 159.9, 162.1, 165.6, 169.3, 170.2. HRMS: m/e
To a solution of 19 (22 mg, 0.042 mmol) and DMAP (1 mg,
0.005 mmol) in CH2Cl2 (0.5 mL) was added triethylamine (0.04
mL, 0.12 mmol) and acetic anhydride (0.03 mL, 0.08 mmol) at
0 °C. The reaction mixture was stirred overnight, quenched with
saturated aqueous NH4Cl solution (20 mL) and extracted with
CH2Cl2 (20 mL ꢂ 3). The organic layer was dried over anhydrous
MgSO4 and solvent was removed under reduced pressure to afford
a liquid residue. The residue was purified by column chromatogra-
phy on silica gel using hexanes/EtOAc (2:1) as the eluent to afford
16c (21 mg, 80% yield) as colorless oil: ½a D22
ꢄ
+5.5 (c 0.5, CHCl3); 1H
NMR (600 MHz, CDCl3) d 1.02 (m, 21H), 1.95 (s, 3H), 2.18 (m, 2H),
3.55 (d, J = 13.2 Hz, 1H), 3.92 (d, J = 17.8 Hz, 1H), 4.04 (d, J = 13.2,
5.4 Hz, 1H), 4.35 (dd, J = 10.2, 6.0 Hz, 1H), 4.39 (d, J = 18.0 Hz,
1H), 4.46 (s, 1H), 4.25 (d, J = 12.6 Hz, 1H), 5.57 (d, J = 12.0 Hz,
1H), 5.59 (s, 1H), 7.19 (d, J = 7.2 Hz, 1H), 7.29 (t, J = 7.2 Hz, 1H),
7.33 (d, J = 7.2 Hz, 1H), 7.43 (t, J = 7.2 Hz, 2H), 7.57 (t, J = 7.8 Hz,
1H), 7.99 (d, J = 7.8 Hz, 2H); 13C NMR (125.7 MHz, CDCl3) d 12.1,
18.1, 29.9, 41.1, 44.3, 57.8, 60.0, 64.8, 67.9, 69.9, 128.9, 129.3,
129.5, 130.0, 130.5, 132.2, 133.9, 134.7, 134.8, 135.5, 165.8,
167.9, 170.5. HRMS: m/e calcd for C32H43NO6SiH+: 566.2938,
calcd for C46H40N2O10H+: 781.2761, found: 781.2789 (
ppm).
D = 3.6
found: 566.2955 (
D
= 3.0 ppm).
6.1.26. (2S,10S,10aR)-9-Acetoxymethyl-10-benzoyloxy-5-oxo-1,
2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-
30-benzoylamino-20-hydroxy-30-phenylpropanoate (1)
6.1.22. (2S,11S,11aR)-10-(Acetoxymethyl)-11-benzoyloxy-2-
hydroxy-2,3,11,11a-tetrahydro-1H-benzo[d]-pyrrolo[1,2-a]-
azepin-5(6H)-one (17c)
To a solution of 21a (26 mg, 0.03 mmol) in methanol (0.5 mL)
was added p-toluenesulfonic acid (1.5 mg, 0.006 mmol). After stir-
ring the mixture overnight, the solvent was removed and the resi-
due purified by column chromatography on silica gel using
hexanes/EtOAc = 1:2 as the eluent to afford 1 as a white solid
White solid; 86% yield; 1H NMR (500 MHz, CDCl3) d 2.00 (s, 3H),
2.20 (m, 2H), 3.64 (d, J = 13.5 Hz, 1H), 3.93 (d, J = 17.5 Hz, 1H), 3.98
(m, 1H), 4.40 (m, 2H), 4.48 (s, 1H), 5.26 (d, J = 12.5, 1H), 5.58 (d,
J = 12.5 Hz, 1H), 6.61 (s, 1H), 7.18 (d, J = 7.0 Hz, 1H), 7.29 (t,
J = 8.0 Hz, 1H), 7.34 (d, J = 7.5 Hz, 1H), 7.45 (t, J = 8.0 Hz, 2H), 7.59
(d, J = 7.5 Hz, 1H), 7.99 (d, J = 7.5 Hz, 2H); 13C NMR (125.7 MHz,
CDCl3) d 20.8, 39.8, 44.0, 57.1, 59.5, 64.5, 67.0, 69.5, 128.7, 129.1,
129.3, 129.7, 129.8, 130.0, 131.8, 133.7, 134.3, 134.5, 135.2,
165.6, 167.9, 170.3. HRMS: m/e calcd for C23H23NO6H+: 410.1604,
(16 mg, 75% yield): mp 116–118 °C; ½a D22
ꢄ
ꢀ135 (c 2.7, CHCl3); 1H
NMR (400 MHz, CDCl3) d 1.90 (s, 3H), 2.37 (td, J = 8.8, 3.2 Hz, 1H),
2.48 (dd, J = 11.2, 4.4 Hz, 1H), 3.18 (d, J = 2.8 Hz, 1H), 3.99 (dd,
J = 11.2, 3.6 Hz, 1H), 4.03 (d, J = 12.0 Hz, 1H), 4.44 (td, J = 8.8,
4.4 Hz, 1H), 4.62 (s, 1H), 5.05 (d, J = 10.0 Hz, 1H), 5.22 (d,
J = 10.4 Hz, 1H), 5.59 (t, J = 3.2 Hz, 1H), 5.69 (d, J = 7.6 Hz, 1H),
6.71 (d, J = 9.2 Hz, 1H), 6.74 (d, J = 7.2 Hz, 1H), 7.17 (t, J = 6.0 Hz,
1H), 7.41 (m, 11H), 7.61 (t, J = 6.0 Hz, 1H), 8.13 (d, J = 6.0 Hz, 2H),
8.20 (dd, J = 5.6, 1.6 Hz, 1H); 13C NMR (100.5 MHz, CDCl3) d 20.3,
38.1, 51.5, 54.4, 58.7, 65.5, 73.2, 73.6, 126.8, 126.9, 128.0, 128.4,
128.5, 128.7, 128.7, 128.9, 128.9, 130.0, 130.3, 131.5, 133.2, 133.6,
133.7, 134.5, 136.0, 138.4, 162.5, 165.7, 166.7, 170.2, 172.2. HRMS:
found: 410.1594 (
D
= ꢀ2.4 ppm).
6.1.23. (4S,5R)-2-(4-Methoxyphenyl)-3-benzoyl-4-phenyloxaz-
olidine-5-carboxylic acid (20a)
White solid; 67% yield; 1H NMR (400 MHz, CDCl3) d 3.82 (s, 3H),
4.41 (s, 1H), 4.90 (s, 1H), 5.48 (s, 1H), 6.85 (d, J = 8.4 Hz, 2H), 6.90
(m, 1H), 7.21–7.39 (m, 11H); 13C NMR (100.5 MHz, CDCl3) d 55.0,
55.6, 72.5, 114.3, 127.0, 127.1, 127.3, 128.1, 128.3, 128.3,
m/e calcd for C38H34N2O9H+: 663.2343, found: 663.2361 (
D = 2.8
ppm).

L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
7111
6.1.27. (2S,10S,10aR)-10-Benzoyloxy-5-oxo-9-pent-4-enoyloxy-
methyl-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-2-yl
(40S,50R)-40-(2-allyloxyphenyl)-30-benzoyl-20-(4-methoxy-
phenyl)oxazolidine-5-carboxylate (21b)
J = 11.0, 5.5 Hz, 1H), 4.65 (dd, J = 4.2, 1.2 Hz, 1H), 5.39 (d,
J = 9.0 Hz, 1H), 5.41 (d, J = 12.5 Hz, 1H), 5.49 (d, J = 12.5 Hz, 1H),
5.74 (d, J = 7.5 Hz, 1H), 6.64 (s, 1H), 6.86 (d, J = 9.0 Hz, 1H), 7.21
(d, J = 8.0 Hz, 1H), 7.26 (m, 4H), 7.43 (m, 6H), 7.55 (m, 3H), 7.75
(d, J = 9.0 Hz, 2H), 7.98 (d, J = 7.5 Hz, 2H); 13C NMR (100.5 MHz,
CDCl3) d 22.6, 36.8, 44.0, 53.7, 54.4, 59.6, 64.2, 69.1, 72.6, 73.1,
126.8, 127.0, 128.0, 128.7, 128.8, 129.0, 129.4, 129.7, 129.8,
131.7, 131.9, 133.7, 133.8, 134.2, 134.5, 135.0, 138.2, 165.4,
166.9, 168.1, 170.5, 172.4. HRMS: m/e calcd for C39H36N2O9H+:
White solid; 96% yield; mp 73-75 °C; ½a D22
ꢄ
-145 (c 2.0, CHCl3);
1H NMR (400 MHz, CDCl3) d 2.27 (m, 5H), 2.42 (m, 1H), 3.81 (s,
3H), 3.86 (d, J = 14.4 Hz, 1H), 4.05 (dd, J = 14.4, 4.8, 1H), 4.25 (td,
J = 10.8, 5.6, Hz, 1H), 4.39 (m, 2H), 4.80 (s, 1H), 4.93 (dd, J = 10.0,
0.8 Hz, 1H), 4.96 (dd, J = 17.6, 1.6 Hz, 1H), 5.03 (d, J = 10.4 Hz,
1H), 5.08 (s, 1H), 5.11 (d, J = 4.8 Hz, 1H), 5.24 (d, J = 12.8 Hz, 1
H0, 5.43 (s, 1H), 5.55 (t, J = 4.8 Hz, 1H), 5.74 (m, 2H), 6.71 (d,
J = 10.4 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.81 (m, 3H), 6.94 (s, 1H),
7.08 (m, 3H), 7.19 (m, 5H), 7.50 (m, 6H), 7.64 (t, J = 7.2 Hz, 1H),
8.13 (d, J = 7.6 Hz, 2H), 8.22 (dd, J = 7.6, 1.6 Hz, 1H); 13C NMR
(100.5 MHz, CDCl3) d 28.5, 32.9, 38.4, 51.9, 55.2, 58.8, 65.2, 68.8,
72.4, 73.0, 111.5, 113.3, 115.5, 117.7, 120.5, 126.9, 128.2, 128.7,
128.7, 128.8, 128.8, 128.9, 129.2, 129.9, 130.2, 132.5, 133.4,
133.9, 134.6, 135.3, 136.5, 154.7, 159.8, 162.0, 165.5, 169.3,
172.3. HRMS: m/e calcd for C52H48N2O11H+: 877.3336, found:
677.2499, found: 677.2502 (
D = 0.4 ppm).
6.1.30. Macrocyclic paclitaxel mimic 3
To a solution of 22 (17 mg, 0.02 mmol) in CH2Cl2 (12 mL) was
added Cl2Ru(=CHPh)(PCy3)2 (3 mg, 0.004 mmol) in CH2Cl2
(0.2 mL). The mixture was stirred at room temperature for 3 days
and reflux for 2 days. The solvent was removed under reduced
pressure. The residue was passed through a short silica gel column
(hexanes/EtOAc = 1:1.5) to remove the catalyst and then afford 3
(8 mg, 66% yield based on 71% conversion) as a white solid, as well
as the starting material (5 mg).
877.3322 (
D
= ꢀ1.6 ppm).
Compound 3: 1H NMR (400 MHz, CDCl3) d 2.30 (m, 5H), 2.78
(dd, J = 10.4, 3.6 Hz, 1H), 3.32 (d, J = 2.8 Hz, 1H), 3.82 (dd, J = 10.0,
1.2 Hz, 1H), 3.96 (m, 2H), 4.36 (dd, J = 11.2, 2.4 Hz, 1H), 4.52 (s,
1H), 4.59 (m, 1H), 4.77 (d, J = 9.2 Hz, 1H), 5.44 (d, J = 9.2 Hz, 1H),
5.56 (s, 2H), 6.14 (dd, J = 7.2, 1.6 Hz, 1H), 6.72 (d, J = 7.6 Hz, 1H),
6.75 (d, J = 9.6 Hz, 1H), 6.78 (d, J = 6.4 Hz, 1H), 6.96 (t, J = 6.4 Hz,
1H), 7.24 (m, 1H), 7.42 (m, 3H), 7.46 (m, 4H), 7.59 (m, 3H), 7.74
(dd, J = 6.0, 1.2 Hz, 1H), 8.21 (dd, J = 6.4, 1.2 Hz, 1H), 8.36 (dd,
J = 6.0, 0.4 Hz, 2H); 13C NMR (100.5 MHz, CDCl3) d 28.2, 33.6,
36.9, 49.2, 51.9, 59.1, 64.1, 68.4, 72.9, 73.0, 73.3, 112.4, 121.0,
125.8, 127.0, 127.5, 128.1, 128.4, 128.5, 128.6, 128.6, 129.2,
129.2, 129.6, 130.5, 130.6, 130.7, 131.6, 131.8, 133.8, 134.0,
137.0, 138.8, 154.8, 162.2, 166.7, 171.8, 172.6. HRMS: m/e calcd
6.1.28. (2S,10S,10aR)-10-Benzoyloxy-5-oxo-9-pent-4-enoyloxy-
methyl-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinolin-2-yl
(20S,30R)-30-(2-allyloxyphenyl)-30-benzoylamino-20-hydroxypro-
panoate] (22)
White solid; 82% yield; mp 110–112 °C; ½a D22
ꢀ139 (c 1.6,
ꢄ
CHCl3); 1H NMR (400 MHz, CDCl3) d 2.27 (m, 6H), 3.34 (br s,
1H), 3.95 (m, 2H), 4.29 (d, J = 8.0 Hz, 1H), 4.33 (d, J = 8.0 Hz,
1H), 4.51 (d, J = 4.8 Hz, 2H), 4.65 (d, J = 3.2 Hz, 1H), 4.92 (dd,
J = 9.2, 1.6 Hz, 1H), 4.96 (dd, J = 17.2, 1.6 Hz, 1H), 5.09 (d,
J = 12.8 Hz, 1H), 5.21 (dd, J = 12.0, 1.2 Hz, 1H), 5.23 (dd, J = 19.6,
12.8 Hz, 1H), 5.48 (dd, J = 25.2, 1.6 Hz, 1H), 5.66 (d, J = 6.0 Hz,
1H), 5.73 (m 1H), 5.98 (m, 2H), 6.67 (d, J = 10.8 Hz, 1H), 6.78 (d,
J = 8.0 Hz, 1H), 6.87 (t, J = 7.2 Hz, 1H), 7.19 (m, 5H), 7.38 (t,
J = 11.6 Hz, 1H), 7.45 (m, 6H), 7.62 (t, J = 7.2 Hz, 1H), 8.13 (d,
J = 7.2 Hz, 2H), 8.18 (dd, J = 7.6, 1.6 Hz, 1H); 13C NMR
(100.5 MHz, CDCl3) d 28.5, 32.9, 38.2, 51.6, 52.0, 58.8, 65.3,
68.7, 72.9, 73.2, 111.8, 115.5, 117.7, 120.9, 126.1, 126.8, 127.8,
128.4, 128.5, 128.7, 128.8, 128.9, 129.2, 130.0, 130.3, 131.4,
132.5, 133.3, 133.7, 133.9, 134.5, 135.9, 136.5, 155.5, 162.3,
165.6, 166.7, 172.1, 172.2. HRMS: m/e calcd for C44H42N2O10H+:
for C42H38N2O10H+: 731.2605, found: 731.2597 (
D
= ꢀ1.1 ppm).
6.1.31. (2S,8S,8aR)-8-(3-Methoxybenzoyloxy-6,7-di-dehydroin-
dolizidin-5-one-2-yl (20R,30S)-20-hydroxy-30-phenyl-30-benzyl-
aminopropionate (24)
White solid; 76% yield for 2 steps; mp 78-80 °C; 1H NMR
(400 MHz, CDCl3)
d 2.09 (td, J = 10.8, 4.4 Hz, 1H), 2.47 (dd,
J = 14.0, 5.6 Hz, 1H), 3.54 (br s, 1H), 3.82 (s, 3H), 3.83 (m, 2H),
4.33 (td, J = 11.2, 5.3, Hz, 1H), 4.64 (d, J = 2.0 Hz, 1H), 5.11 (d,
J = 3.6 Hz, 1H), 5.75 (m, 2H), 6.01 (dd, J = 10.4, 2.8 Hz, 1H), 6.53
(dd, J = 10.0, 1.6 Hz, 1H), 7.13 (dd, J = 8.0, 2.0 Hz, 1H), 7.19-7.41
(m, 6H), 7.45 (d, J = 7.6 Hz, 2H), 7.55 (d, J = 7.2 Hz, 1H), 7.59 (t,
J = 2.0 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H); 13C NMR (100.5 MHz, CDCl3)
d 31.5, 38.2, 50.8, 54.5, 55.4, 55.4, 58.3, 73.0, 114.4, 120.2, 122.5,
125.7, 126.9, 126.9, 128.0, 128.5, 128.8, 129.6, 130.3, 131.6,
133.8, 140.6, 159.6, 162.4, 165.7, 166.8, 172.2. HRMS: m/e calcd
759.2918, found: 759.2893 (
D
= ꢀ3.3 ppm).
6.1.29. (2S,11S,11aR)-10-(Acetoxymethyl)-11-benzoyloxy-2-hy-
droxy-2,3,11,11a-tetrahydro-1H-benzo[d]-pyrrolo-5(6H)-oxo-
[1,2-a]azepin-2-yl (20S,30R)-30-benzoylamino-20-hydroxy-30-
phenylpropanoate (2)
To
a solution of 17c (11 mg, 0.027 mmol), 20a (12 mg,
0.03 mmol) and DMAP (3.2 mg, 0.027 mmol) in CH2Cl2 (0.5 mL)
was added EDC (22 mg, 0.11 mmol) and the mixture was stirred
for 1 day. The reaction mixture was then quenched with EtOAc
(50 mL) and washed with water (10 mL ꢂ 2) and brine (10 mL).
The organic layer was dried over anhydrous MgSO4, filtered, and
then solvent was removed under reduced pressure. The residue
was purified by column chromatography on silica gel using hex-
anes/EtOAc (1:1) as the eluent to afford the coupling product 21c
as a white solid (11 mg, 92% based on 50% conversion).
To a solution of 21c (11 mg, 0.014 mmol) in methanol (0.5 mL)
was added p-TSA (0.5 mg, 0.003 mmol). After stirring overnight,
the solvent was removed and the residue purified by column chro-
matography on silica gel using hexanes/EtOAc (1:2) as the eluent
to afford 2 as a white solid (7 mg, 74% yield for 2 steps): mp
130-132 °C; 1H NMR (500 MHz, CDCl3) d 1.95 (s, 1H), 2.27 (td,
J = 11.0, 5.0 Hz, 1H), 2.40 (dd, J = 13.5, 6.0 Hz, 1H), 3.27 (d,
J = 4.0 Hz, 1H), 3.79 (d, J = 14.0 Hz, 1H), 4.02 (d, J = 17.0, 1H),
4.04 (dd, J = 14.5, 5.0 Hz, 1H), 4.40 (dd, J = 17.0 Hz, 1H), 4.50 (dd,
for C32H30N2O8Na+: 593.1900, found: 593.1893 (
D
= ꢀ1.2 ppm).
6.2. In vitro cell growth inhibition assay
Tumor cell growth inhibition was determined according to the
method established by Skehan et al.34 Human cancer cell lines,
LCC6-WT (Pgpꢀ), MCF-7 (Pgpꢀ), LCC6-MDR (Pgp+), NCI/ADR
(Pgp+), A2780 (Pgpꢀ) and HT-29 (Pgpꢀ) were plated at a density
of 400–2000 cells/well in 96-well plates and allowed to attach
overnight. These cell lines were maintained in RPMI-1640 medium
supplemented with 5% fetal bovine serum and 5% Nu serum (Col-
laborative Biomedical Product, MA). Paclitaxel mimics were dis-
solved in DMSO and further diluted with RPMI-1640 medium.
Triplicate wells were exposed to various treatments. After 72 h
incubation, 100 lL of ice-cold 50% trichloroacetic acid (TCA) was
added to each well, and the samples were incubated for 1 h at
4 °C. Plates were then washed five times with water to remove

7112
L. Sun et al. / Bioorg. Med. Chem. 18 (2010) 7101–7112
TCA and serum proteins, and 50
l
L of 0.4% sulforhodamine B (SRB)
ing preliminary results on the TR-NOESY analysis for binding of mi-
mic 2 to tubulin, as well as the determination of the critical
concentration of tubulin for polymerization in the presence of mi-
mic 2. L.S. and I.O. also thank Ms. Rebecca Rowehl for her valuable
help at the Cell Culture and Hybridoma Facility at State University
of New York at Stony Brook.
was added to each well. Following a 5 min incubation, plates were
rinsed five times with 0.1% acetic acid and air-dried. The dye was
then solubilized with 10 mM Tris base (pH 10.5) for 5 min on a
gyratory shaker. Optical density was measured at 570 nm. The
IC50 values were then calculated by fitting the concentration-effect
curve data with the sigmoid-Emax model using nonlinear regres-
sion, weighted by the reciprocal of the square of the predicted
effect.35
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.069.
6.3. Computational methods
6.3.1. Construction of molecular complexes
References and notes
Paclitaxel mimics 1 and 2 were manually docked into the pac-
litaxel binding site of the REDOR-Taxol-1JFF structure18,19 using
the InsightII 2000 program (CVFF force field) by overlaying three
hydroxyl groups of each mimic with those of the paclitaxel mole-
cule (C13, C2, and C4 hydroxyl groups) with the REDOR-Taxol-
ITUB13,18 conformation. The resulting molecular complex was
energy-minimized in 5000 steps or until the maximum derivative
became <0.001 kcal/Å by means of the conjugate gradients method
using the CVFF force field and the distance-dependent dielectric.
The backbone of the protein was fixed during the energy minimi-
zation. In the same manner, the molecular complex of paclitaxel
mimic 3 was obtained. The overlays of mimics 1, 2 and 3 with RE-
DOR-Taxol-1TUB are shown in Figure 3.
1. Jemal, A.; Ward, E.; Hao, Y.; Thun, M. J. Am. Med. Assoc. 2005, 294, 1255.
2. Jemal, A.; Siegel, R.; Ward, E.; Murray, T.; Xu, J.; Smigal, C.; Thun Michael, J. CA
Cancer J. Clin. 2006, 56, 106.
3. Rowinsky, E. K. Ann. Rev. Med. 1997, 48, 353.
4. Suffness, M. E. Taxol Science and Applications; CRC Press: Boca Raton, 1995. p
426.
5. Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277, 665.
6. Schiff, P. B.; Horwitz, S. B. Proc. Natl Acad. Sci. U.S.A. 1980, 77, 1561.
7. Ojima, I.; Wang, T.; Delaloge, F. Tetrahedron Lett. 1998, 39, 3663.
8. Kingston, D. G. I. J. Nat. Prod. 2000, 63, 726.
9. Kingston, D. G. I. Chem. Commun. 2001, 867.
10. Ojima, I.; Chen, J.; Sun, L.; Borella, C. P.; Wang, W.; Miller, M. L.; Lin, S.; Geng, X.;
Kuznetsova, L.; Qu, C.; Gallager, D.; Zhao, X.; Zanardi, I.; Xia, S.; Horwitz, S. B.;
Mallen-St. Clair, J.; Guerriero, J. L.; Bar-Sagi, D.; Veith, J. M.; Pera, P.; Bernacki, R.
J. J. Med. Chem. 2008, 51, 3203.
11. Ojima, I.; Das, M. J. Nat. Prod. 2009, 72, 554.
The molecular complexes of 1 and 2 in the 1JFF tubulin32 were
constructed using the same protocol as that described above except
for employing REDOR-Taxol-1JFF18,19 in place of REDOR-Taxol-
1TUB. Since there are differences in the protein structure between
1TUB and 1JFF, the mimics’ structures underwent small changes,
but the critical H-bond between the C20-OH and His229 was very
stable during the energy minimization. After the energy minimiza-
tion, the snapshots were overlaid by superimposing the backbones
of the proteins. The overlays are shown in Figure 5. The mimics 1
and 2 showed very good overlays with REDOR-Taxol-1JFF.
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6.3.2. MD simulations of mimics 1 and 2 in 1JFF
To examine the stability of the structures of mimics 1 and 2,
molecular dynamics (MD) simulation was performed using the
Macromodel program (MMFF94 force field).33 The molecular com-
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for the MD simulations with a 10 Å sphere around the binding site
at 300 K with the time step of 0.5 fs for 50 ps in a generalized Born
with surface area term (GBSA) continuum solvent description of
water solvation.36 Within the 10 Å sphere of the binding site, the
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This work was supported by grants from the National Institutes
of Health (CA103314 and GM42798 to I.O.; CA 73872 to R.J.B.). The
authors are grateful to Dr. J. Fernando Díaz and his laboratory at
the Centro de Investigaciones Biológicas, Madrid, Spain for obtain-