Design and synthesis of simplified taxol analogs based on the T-Taxol bioactive conformation

Article abstract of DOI:10.1016/j.bmc.2011.10.010

A series of compounds designed to adopt a conformation similar to the tubulin-binding T-Taxol conformation of the anticancer drug paclitaxel has been synthesized. Both the internally bridged analogs 37-39, 41 and the open-chain analogs 27-29 and 43 were prepared. The bridged analogs 37-39 and 41 were synthesized by Grubbs' metatheses of compounds 30-32 and 33, which, in turn, were prepared by coupling β-lactams 24-26 with alcohols 22 and 23. Both the bridged and the open-chain analogs showed moderate to good cytotoxicity.

Full text of DOI:10.1016/j.bmc.2011.10.010

Bioorganic & Medicinal Chemistry 19 (2011) 7664–7678
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of simplified taxol analogs based on the T-Taxol
bioactive conformation
Jielu Zhao a, Susan Bane b, James P. Snyder c, Haipeng Hu c, Kamalika Mukherjee b,
Carla Slebodnick a, David G.I. Kingston a,
⇑
a Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, USA
b Department of Chemistry, State University of New York at Binghamton, Binghamton, NY 13902, USA
c Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of compounds designed to adopt a conformation similar to the tubulin-binding T-Taxol confor-
mation of the anticancer drug paclitaxel has been synthesized. Both the internally bridged analogs
37–39, 41 and the open-chain analogs 27–29 and 43 were prepared. The bridged analogs 37–39 and
41 were synthesized by Grubbs’ metatheses of compounds 30–32 and 33, which, in turn, were prepared
by coupling b-lactams 24–26 with alcohols 22 and 23. Both the bridged and the open-chain analogs
showed moderate to good cytotoxicity.
Received 10 August 2011
Revised 30 September 2011
Accepted 5 October 2011
Available online 13 October 2011
Keywords:
Simplified paclitaxel
T-Taxol
Ó 2011 Elsevier Ltd. All rights reserved.
Bioactive conformation
Taxol
Paclitaxel
X-ray
Modeling
Tubulin
Antiproliferative activity
R2O
OR3
O
1. Introduction
O
R1
NH
O
The clinically important naturally occurring anticancer agent
paclitaxel (TaxolÒ) (1a) was first isolated in the 1960’s by Wall
and Wani, and its structure was published in 1971.1 After a difficult
and lengthy development period2 it was approved in 1992 by the
US Food and Drug Administration (FDA) for the treatment of ovar-
ian cancer and in 1994 for treatment of breast cancer. The drug, to-
gether with its semisynthetic analog docetaxel (1b),3 have become
two of the most important and widely used anticancer drugs for
treatment of breast4 and ovarian cancers.5 Their superior biological
activities have spurred extensive studies on the chemistry and
structure–activity relationships of the taxanes, and virtually every
position of the molecule has been modified.6 Numerous synthetic
analogs with better activities and pharmacokinetic properties have
been developed and some promising analogs have entered clinical
trials.7 However, none of the analogs other than docetaxel (1b)8
and cabazitaxel (1c)9 have yet been approved for clinical use by
the Food and Drug Administration.
O
H
Ph
O
AcO
HO
OH
OCOPh
1a R1 = Ph, R2 = Ac, R3 = H
1b R1 = (CH3)3CO, R2 = R3 = H
1c R1 = (CH3)3CO, R2 = R3 = CH3
Paclitaxel exerts its anticancer activity by binding to the protein
tubulin and promoting its polymerization to microtubules.10 The
disruption of the normal tubulin-microtubule dynamics leads to
abnormal cell division followed by apoptosis.11 The interaction of
paclitaxel with tubulin has been studied intensively by several
methods, including photoaffinity labeling,12 fluorescence spectros-
copy13 and electron crystallography.14 While these studies pro-
vided crucial information on the nature of the paclitaxel–tubulin
interaction, only the latter has been able to furnish details of the
actual binding conformation of paclitaxel.
⇑
Corresponding author. Tel.: +1 540 231 6570; fax: +1 540 231 3255.
E-mail address: dkingston@vt.edu (D.G.I. Kingston).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.010

J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
7665
Early investigations of the conformation of paclitaxel in solution
O
led to the identification of both ‘polar’15 and ‘non-polar’16 confor-
mations, based on NMR studies of paclitaxel in polar and non-polar
solvents, respectively. Later NMR studies using the NAMFIS analysis
identified the T-Taxol conformation as a third solution-phase con-
formation,17 and this was proposed to be the tubulin-binding con-
formation by Snyder and coworkers based on computational
studies and data obtained from the electron density map of paclit-
axel-bound tubulin from the electron crystallography studies.18 The
validity of the T-Taxol model was then established experimentally
by the determination of intramolecular distances in tubulin-bound
paclitaxel by REDOR NMR experiments19 and by the synthesis of
constrained paclitaxel analogs such as 2. This compound adopts
the T-Taxol conformation and is significantly more active than pac-
litaxel in both cytotoxicity and tubulin polymerization assays.20
Analog 2, in its 3-D representation, thus defines the required con-
formation for the effective binding of paclitaxel analogs to tubulin.
O
HO
N
H
N
H
HO
O
Ph
N
H
OH
O
OBz
OH
bridge
Figure 1. General structure of second generation tricyclic core (left) and T-Taxol
mimics (right).
However, it was found to be highly insoluble in water, which made
its biological evaluation very difficult, and it had only modest tubu-
lin assembly activity.23
In this second approach to the objective of developing a new
generation of water-soluble simplified paclitaxel analogs, an aza-
tricyclic moiety (Fig. 1, left) was designed to mimic the baccatin
core of paclitaxel, based on an earlier bicyclic analog prepared by
Ojima.22a The basic tertiary nitrogen was anticipated to increase
water-solubility, and the aza-tricyclic moiety is functionalized
with three hydroxyl groups where the key side chains can be at-
tached. The final construct (Fig. 1, right) can be constrained to
the T-Taxol conformation by selection of an appropriate length
for the bridge linking the side chain and the tricyclic core. This
new core bears three stereogenic centers which can lead to eight
possible stereoisomers. The availability of various hydroxyprolines
as starting materials allows for control of two of these three
centers.
A computer modeling study was performed to determine which
of the eight possible isomers best serves as the optimal replace-
ment of the baccatin structure. Thus, a conformational search
was carried out for each of the eight isomers using the MMFF force
field embedded in Maestro,24 and subsequently each of the eight
global minima was fitted with analog side chains and various
bridges. Each such structure was then geometry optimized and
Glide docked25 into the paclitaxel binding site on tubulin. Individ-
ual dockings returned a variety of poses (i.e. binding modes). Those
that best mimicked the shape and extension of 2 were selected and
evaluated with the independent MM-GBSA scoring function.26
Figure 2 shows the two most favorable binding structures (4 and
5) together with the active analog 2 in a 3-D representation of
the tubulin binding site. Compounds 4 and 5 are derived from
two different stereoisomers of the aza-tricyclic moiety, with
S- and R-configurations at C-10, respectively.
AcO
O
OH
O
HO
O
O
O
H
OBz
HO
O
O
Ph
N
H
2
The synthesis of paclitaxel is a challenging task, and although
several outstanding total syntheses have been published,21 none
of them are scaleable to economical commercial production. The
synthesis of structurally simplified paclitaxel analogs with retained
or even improved activities is thus an attractive goal. Although sev-
eral attempts toward this goal have been reported,22 the analogs
resulting from these studies were either inactive or significantly
less active than paclitaxel.
O
HO
O
O
O
BzO
N
H
Ph
O
O
3
Our group has previously designed and synthesized the simpli-
fied paclitaxel analog (3) where a 2.2.1-bicyclononane moiety was
used as a structurally simpler surrogate of the baccatin core.23 The
logic behind this design was that the northern hemisphere of the
paclitaxel structure is less crucial to its bioactivity based on its
known SAR,6a and that a structure with the T-Taxol pharmaco-
phore could be achieved by installation of the paclitaxel side chain
to a simple bridged bicyclic skeleton and subsequent constraint of
the conformation by a suitable bridge. The resulting compound 3
showed moderate activity in assays for antiproliferative activity.
Encouraged by these results we elected to prepare analogs aris-
ing from both stereoisomers and with different bridge lengths for
comparison. For easier synthetic manipulation, the bridged struc-
ture was modified to that of 6, containing an ether linkage rather
than the ester linkage of compounds 4 and 5. After the synthetic
part of this work had been completed, Ojima reported the synthe-
sis and biological evaluation of a similar series of compounds, al-
beit with longer bridges and other variations.22b The present
work can be viewed as an extension of this parallel study.
O
O
O
HO
N
H
N
H
HO
HO
N
O
O
O
O
O
O
R
S
Ph
N
H
Ph
N
H
Ph
N
H
H
n
OBz
OBz
X
O
O
OBz
O
O
O
4
5
6
X = O or bond
n = 1-3

7666
J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
Figure 2. Left: low energy poses of compounds of 4 (in green) and 2 (in yellow) in the paclitaxel tubulin binding site. Right: low energy poses of compounds 5 (in green) and 2
(in yellow) in the paclitaxel tubulin binding site.
be unstable on a silica gel column, and so it was reduced directly
with sodium borohydride to afford 20 as the only stereoisomer
in an overall yield of 42% for three steps. The stereoselectivity
was conceivably driven by the bulky TBS protecting group. The
absolute stereochemistry of 20 was determined by X-ray crystal-
lography of compound 21 (Fig. 3), which was prepared by acylation
of the newly generated hydroxyl group followed by deprotection of
the silyl ether. As expected, reduction of 19 after deprotection of
the large protecting group produced the epimeric diol 22 as the
major product, together with lesser amounts of diol 23
(22:23 = 3:2). The absolute stereochemistry of the former was also
determined by X-ray crystallography (Fig. 3).
The known b-lactams 24–26 were prepared following the liter-
ature procedures.30 They were coupled with alcohol 21 to generate
open-chain analogs 27–29 in good yields (Scheme 5). The subse-
quent ring closing metatheses of these alkenes with Grubbs’ 2nd
generation catalyst failed to bring about the desired bridged prod-
ucts, even though several different catalysts and conditions were
evaluated. Examination of the 3-D structure of a low energy con-
former of compound 28 (Fig. 4) suggested that the benzoyl group
interposing between the two olefins could be responsible for the
low reactivity. Thus, an alternate synthesis was developed allowing
introduction of the benzoyl group after alkene metathesis.
2. Results and discussion
The retro-synthetic analysis of compounds of general structure
6 is shown in Scheme 1. The synthesis of the simplified analogs
started with the construction of the aza-tricyclic core from
cis-4-hydroxyproline derivative 7 and aldehyde 8, followed by its
coupling with the b-lactam forms of the side chain derivatives to
furnish the side chains and finally Grubbs’ metatheses to furnish
the bridges.
The cis-4-hydroxyproline derivative 7 was synthesized from
commercially available trans-4-hydroxy-L-proline in nine straight-
forward steps with high yields (Scheme 2). The amino acid was
protected as its carbamate and methyl ester derivative 9,27 fol-
lowed by a Mitsunobu reaction, which successfully inverted the
stereochemistry at C-4.28 The resulting ester 10 was hydrolyzed
and reprotected as its TBS ether 11 with an overall yield of 63%
in five steps. Compound 11 was hydrolyzed to acid 12, which
was converted to amide 14 in two steps. The Boc protecting group
was selectively removed with ZnBr2 to produce 7 in 95% yield.
Aldehyde 8 was synthesized from 3-methoxybenzyl alcohol in
four steps (Scheme 3). Commercially available 3-methoxybenzyl
alcohol was treated with butyl lithium and iodine to afford com-
pound 15,29 which was oxidized with Jones’ reagent. The resulting
aldehyde 16 was treated with boron tribromide to cleave the
methyl ether and then combined with allyl bromide in the pres-
ence of base to generate compound 8.
Alkenes 30–32 were prepared by coupling alcohol 23 with
b-lactams 24–26, respectively, and 33 was synthesized from alco-
hol 22 and b-lactam 26 in good yield. Pleasingly, the ring closing
metathesis of alkene 30 gave the desired bridged product, but only
in trace amounts. Since it has been reported that some nitrogen
containing compounds, especially amines, are poor substrates for
Grubbs’ ruthenium catalysts,31 we surmised that the low yield
Reductive amination between compounds 7 and 8 with sodium
triacetoxyborohydride went smoothly, generating compound 18
(Scheme 4). Cyclization of 18 mediated with n-BuLi afforded the
key aza-tricyclic intermediate 19. This compound was found to
reductive amination
N
HO
NH
Holton–Ojima side
chain synthesis
TBSO
H
N
O
OH
Ph
O
butyl lithium
mediated
O
HO
N
7
O
O
ring formation
O
I
Ph
N
H
H
X
OBz
O
n
H
TIPSO
Grubbs'
metathesis
X
O
n
N
O
O
X = O or bond
n = 1 or 2
Ph
X = O or bond
n = 1 or 2
8
Scheme 1. Retro-synthesis of simplified paclitaxel analogs.

J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
COOMe
7667
COOH
NH
COOMe
Boc
COOMe
Boc
COOH
Boc
i, ii
iii
iv, v
vi
N
N Boc
N
N
HO
AcO
HO
vii
TBSO
TBSO
12
9
10
11
CONMePh
CONHPh
CONMePh
NH
O
O
viii
ix
N
O
N
O
TBSO
TBSO
TBSO
14
13
7
Scheme 2. Synthesis of intermediate 7. Reagents and conditions: (i) aqueous NaOH, di-tert-butyl dicarbonate, THF/H2O, overnight; (ii) Cs2CO3, MeI, DMF, overnight; (iii)
AcOH, PPh3, DIAD, THF, overnight; (iv) K2CO3, MeOH, 1 h; (v) TBSCl, imidazole, DMF, 16 h, 63% for five steps; (vi) aqeous LiOH, MeOH, 45 °C, 6 h, 95%; (vii) TEA, ClCOOCH3,
0 °C, TFH, 1 h; aniline, THF, 1 h, 79%; (viii) NaH, MeI, DMF, 93%; (ix) ZnBr2, CH2Cl2, 95%.
O
I
O
I
O
I
HO
HO
i
ii
H
H
H
iii
iv
I
OMe
OMe
15
OMe
O
OH
16
8
17
Scheme 3. Synthesis of compound 8. Reagents and conditions: (i) n-BuLi, I2, hexane/ether, ꢀ78 °C, 57%; (ii) Jones reagent, acetone/water; (iii) BBr3, CH2Cl2, 41% for two steps;
(iv) allyl bromide, K2CO3, DMF, 96%.
OTBS
O
N
H
N
N
TBSO
TBSO
c
a
N
b
7
+
8
Ph
H
O
O
OH
O
19
I
20
7
, 42% from
O
18
N
H
N
N
H
HO
e, c HO
HO
d, e
19
20
H
OH
O
OH
O
OBz
O
23
, 20% from
7
22
, 29% from
7
21
20
, 59% from
Scheme 4. Synthesis of alcohols 21–23. Reagents and conditions: (a) NaBH(OAc)3, ClCH2CH2Cl; (b) BuLi, THF, ꢀ78 °C; (c) NaBH4, MeOH; (d) LiHMDS, PhCOCl, THF; (e)
HFꢁpyridine, THF.
was due to the presence of the tertiary amine in 30. To overcome
this problem, compound 30 was protonated with 10-camphorsul-
fonic acid before the addition of the Grubbs’ catalyst, and the yield
of the metathesis reaction was found to be greatly improved. The
resulting macrocyclic compound was benzoylated and deprotected
to afford the target analog 37 with an overall yield of 61% in three
steps from 30. Compound 38 with a shorter bridge was prepared
from alkene 31 by similar procedures, although the overall yield
was much lower.
In the case of compound 32, the metathesis reaction suffered
from very poor yield (7% for 2 steps), affording the bridged analog
39. Interestingly, the C-2 epimeric alkene 33 reacted smoothly un-
der the same ring closing metathesis conditions to deliver 5-atom
bridged 41 in 33% overall yield from compound 33. The different
reactivities of 32 and 33 were probably due to the opposite orien-
tation of the free hydroxyl groups. The corresponding open-chain
analog 43 was synthesized in two steps from 33.
3. Biological evaluation
The open-chain analogs 27–29, 43 and bridged analogs 37–39,
41 were evaluated for their antiproliferative activities against the
A2780 cell line. The results are summarized in Table 1. The simpli-
fied analogs were cytotoxic, but over 300-fold less cytotoxic than
paclitaxel. The bridged analogs (37–39) exhibited slightly better
activity than their open-chain counterparts 27–29. Compound 41
was significantly more active (4-fold) than the open-chain analog
43.
The analogs were also evaluated for tubulin assembly or disas-
sembly activity with pure
a,b-tubulin. Relatively high concentra-
tions of ligand are required for this type of assay,32 so the
solubility of each molecule as a function of analog concentration
and DMSO concentration was first assessed. The limit of solubility
of these molecules in 10% DMSO in buffer is about 60
bination of weak activity and low solubility severely complicates
lM. The com-

7668
J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
Figure 3. Anisotropic displacement ellipsoid drawing of the X-ray crystal structures of 21 (left) and 22 (right).
O
HO
O
N
TIPSO
O
X
O
a,d
Ph
N
H
H
n
N
X
n
OBz
O
21
+
O
27
28
29
21
24
X = O, n = 2, 61% from
X = O, n = 1, 81% from
X = O, n = 2
21
25 X = O, n = 1
21
X = bond, n = 1, 72% from
26 X = bond, n = 1
O
O
TIPSO
RO
N
N
H
TIPSO
O
O
O
O
b, c
X
O
a
N
n
Ph
N
H
H
n
X
Ph
N
H
X
OH
O
O
+
OBz
O
23
n
34 X = O, n = 2, R = TIPS
35 X = O, n = 1, R = TIPS
30
31
32
,
X = O n = 2, 75%
X = O, n = 1, 89%
24
X = O, n = 2
25 X = O, n = 1
26 X = bond, n = 1
36
X = bond, n = 1, R = TIPS
X = bond, n = 1, 92%
d
37 X = O, n = 2, R = H; 61% from 30
38 X = O, n= 1, R = H; 17% from 31
39
32
X = bond, n = 1, R = H; 7% from
O
O
TIPSO
O
TIPSO
N
H
N
O
RO
O
O
b,c
N
a
O
Ph
N
H
H
Ph
N
H
OBz
22
+
O
O
OH
O
33
26
40 R = TIPS
41 R = H, 33% from 33
c
d
O
RO
N
H
O
O
Ph
N
H
OBz
O
42 R = TIPS
43 R = H, 71% from 33
d
Scheme 5. Synthesis of open-chain and bridged paclitaxel analogs. Reagents and conditions: (a) NaH, THF, 0 °C, 76–92%; (b) 10-camphorsulfonic acid, CH2Cl2, 40 °C; Grubbs’
2nd generation catalyst, CH2Cl2, 40 °C; (c) LiHMDS, benzoyl chloride, ꢀ78 °C, THF; (d) HFꢁPy, THF, 0 °C to rt.
measuring the effect of the molecules on tubulin assembly. A num-
ber of experimental conditions to assess polymerization were eval-
uated, two of which are described.
Figure 5 illustrates a microtubule disassembly assay. In this
assay, the ability of a ligand to stabilize microtubules against
cold-induced depolymerization is assessed. Tubulin is polymerized
in the presence and absence of the ligand by raising the tempera-
ture of the solutions to 37 °C. The concentration of microtubules is
proportional to the amount of light scattered, assessed as the
change in the optical density of the sample at 350 nm. For each

J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
7669
0.8
0.6
0.4
0.2
0.0
0
1000
2000
3000
4000
Figure 4. 3-D structure of 28 optimized with PM3 method in Spartan.
Time (seconds)
sample, after a steady state is reached the temperature of the cell
holder is dropped to 4 °C (arrow, Fig. 5). In the absence of a stabi-
lizing ligand, microtubules are completely disassembled after the
sample is cooled (solid red curve in Fig. 5). A stabilizing ligand such
as paclitaxel increases the concentration of microtubules formed at
37 °C (solid black curve, Fig. 5) and prevents complete disassembly
at 4 °C. A new steady state of microtubule polymers is established
in the paclitaxel-containing sample.
Figure 5. Tubulin assembly was monitored by optical density at 350 nm at 37 °C as
described under Section 5. At the time indicated by the arrow the temperature was
decreased to 4 °C to monitor temperature induced disassembly. Taxol: solid black
line. DMSO only (no ligand): solid red line. Open chain compounds (solid lines): 27
(green), 28 (blue), 29 (pink), 43 (orange). Bridged compounds (dashed lines): 37
(green), 38 (blue), 39 (pink), 41 (orange). The concentration of Taxol was 15
The concentration of all synthetic ligands was 60 M. The concentration of tubulin
was 15 M.
lM.
l
l
The activity of the open chain and bridged analogs were similar
to paclitaxel in this assay. Some of the analogs appeared to increase
the concentration of microtubules and provide some protection
against cold-induced depolymerization, as judged by the higher
plateau values at 37 and 4 °C relative to the control.
30000
20000
10000
0
It was noted that ligand solutions that contain undissolved
material scatter light in the same wavelength range as microtu-
bules. We suspect this to be due to the aggregation of the highly
hydrophobic simplified taxanes.33 Such clusters not only inhibit
proteins in vitro at micromolar concentrations, but also appear to
promote partial protein unfolding.34 Therefore the light scattering
assay cannot distinguish between a solution containing partially
perturbed microtubules and one in which an insoluble and possi-
bly aggregated taxane analog is also present. Also, since the depo-
lymerization assay involves changing the temperature of the
solution, this may also affect the solubility of the ligand. Multiple
parameters of the assay were changed in attempts to verify the
apparent effect of the analogs on microtubule assembly, but the re-
sults of all experiments were ambiguous.
00:00:00
00:20:00
00:40:00
Time (hh:mm:ss)
01:00:00
01:20:00
An alternative method for monitoring tubulin assembly is to ob-
serve the fluorescence of DAPI (40,6-diamidino-2-phenylindole),
which increases as tubulin polymerizes.35 Light scattering from
insoluble material is therefore not directly observed in this detec-
tion method. Again, a number of parameters were varied. Figure 6
shows the results of one of these experiments. The assay condi-
tions were adjusted such that the tubulin concentration was just
slightly below the critical concentration of the protein, so no signif-
icant polymerization is observed in the control (compare red curve
to black curve in Fig. 6). In contrast to the light scattering results,
none of the analogs showed activity in the fluorescence assays.
These experiments illustrate that great care must be taken to
assess molecules that may be weakly active in microtubule assem-
bly assays. Conclusions based on just one or two measurements
Figure 6. Tubulin assembly was monitored by the fluorescence of microtubule-
bound DAPI as function of time at 37 °C using SynergyMx multi-mode
microplate reader as described under Section 5. Taxol (final concentration 8 M) or
test compound (final concentration 60 M) in DMSO (final concentration 10% v/v)
was added to initiate polymerization in solutions containing 8 M tubulin and
10 M DAPI. Taxol: solid black line. DMSO only (no ligand): solid red line. Open
a
a
l
l
l
l
chain compounds (solid lines): 27 (green), 28 (blue), 29 (pink), 43 (orange). Bridged
compounds (dashed lines): 37 (green), 38 (blue), 39 (pink), 41 (orange).
may incorrectly attribute microtubule assembly activity to poorly
soluble and potentially aggregated small molecules, especially
when using light scattering methods. Such an outcome might like-
wise have influenced recent measurements on a related series of
truncated taxanes with cytotoxic activities similar to those re-
ported here (IC50 6 to >20 l
M).22b,33 Unless parallel microtubule
Table 1
Antiproliferative activities (IC50, l
M)a of paclitaxel and the simplified paclitaxel analogs
Compound#
A2780b
37
4.5 0.7
27
38
28
39
29
41
5.1 0.7
43
Paclitaxel
5.8 0.5
4.1 0.4
8.3 0.7
4.0 1.5
4.7 0.6
23.8 5.2
0.015 0.001
a
The concentration at which the compound inhibits 50% of the cell growth.
Human ovarian cancer cell line.
b

7670
J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
assembly measurements are carried out, however, a direct compar-
ison cannot be made.
packed with CeliteÒ. The filtrate was concentrated under reduced
pressure and the residue was partitioned between saturated aque-
ous NaHCO3 solution (50 mL) and EtOAc (50 mL). The aqueous
phase was further extracted with EtOAc (2 ꢂ 50 mL). The com-
bined organic layers were washed, dried and concentrated. Column
chromatography (50% EtOAc in hexanes) afforded the title
4. Conclusions
The simplified paclitaxel analogs 27–29, 37–39, 41 and 43 con-
taining an aza-tricyclic moiety have been designed and synthe-
sized. The bridged analogs 37–39 designed to adopt the T-Taxol
conformation show only very slightly better activities compared
to their open-chain counterparts in the cytotoxicity assay. How-
ever, their low solubility and putative aggregation precluded con-
firmation of microtubule activity in assays using pure tubulin.
The lack of activity relative to paclitaxel in the cells can, in part,
be attributed to the complex conformational profile of the macro-
cyclic rings relative to the parent molecule and to its bridged ana-
logs and, perhaps, in part to micelle-like small molecule clusters
formed both in solution and in cells. These problems are not insur-
mountable, and the synthesis of analogs with modified structures
that can better mimic the T-Taxol conformation based on modeling
studies may yet be achieved by suitable synthetic design.
compound as a yellowish oil (9.5 g, 97% for two steps). ½a D23
ꢃ
ꢀ69.4 (c 1.3, MeOH); 1H NMR (400 MHz) d 4.48 (s, 1H), 4.44
(t, J = 7.7 Hz, 0.4H), 4.38 (t, J = 8.0 Hz, 0.6H), 3.72 (d, J = 4.7 Hz,
3H), 3.61 (dt, J = 15.6, 7.8 Hz, 1H), 3.55 (d, J = 11.7 Hz, 0.6H), 3.44
(d, J = 11.3 Hz, 0.4H), 2.28 (m, 1.6H), 2.16 (s, 0.4H), 2.11–2.00
(m, 1H), 1.75 (s, 0.6H), 1.42 (m, 9H); 13C NMR (126 MHz) d
173.73, 173.51, 154.61, 154.05, 80.47, 80.38, 77.36, 77.31, 77.11,
76.85, 70.31, 69.57, 57.98, 57.55, 54.84, 54.78, 52.35, 52.14,
39.21, 38.56, 28.46, 28.33, 0.07; HRMS (ESI) calcd for C11H20NO5
m/z 246.1341 ([M+H]+), found m/z 246.1333.
5.1.3. (2S,4S)-N-Boc-4-acetoxy-proline methyl ester (10)
To a solution of 9 (9.5 g, 38.7 mmol), triphenyl phosphine (21 g,
80 mmol, 2.1 equiv) and acetic acid (4.6 mL, 80 mmol, 2.1 equiv) in
THF was added diisopropyl azodicarboxylate (DIAD) dropwise
(15.8 mL, 80 mmol, 2.1 equiv) at 0 °C. The resulting mixture was
warmed up to rt and stirred overnight. The solvent was evaporated
under reduced pressure and the residue was purified by column
chromatography (1:2 EtOAc–hexanes) to yield 10 as a colorless
5. Experimental section
5.1. Chemistry
oil (9.7 g, 87%). ½a D23
ꢃ
ꢀ18.4 (c 1.1, MeOH); 1H NMR (500 MHz) d
5.1.1. General experimental methods
5.23–5.17 (m, 1H), 4.48 (d, J = 9.0 Hz, 0.5H), 4.36 (d, J = 9.2 Hz,
0.5H), 3.75–3.66 (m, 4H), 3.57 (d, J = 12.4 Hz, 0.5H), 3.49 (d,
J = 12.4 Hz, 0.5H), 2.43 (m, 1H), 2.31–2.20 (m, 1H), 1.98 (s, 3H),
1.41 (m, 9H); 13C NMR (126 MHz) d 172.63, 172.28, 170.50,
170.33, 154.18, 80.40, 72.94, 71.79, 62.63, 57.81, 57.43, 52.32,
52.16, 51.82, 36.37, 35.43, 28.44, 28.33; HRMS (ESI) calcd for
The following standard conditions apply unless otherwise sta-
ted. All reactions were performed under argon or nitrogen in
oven-dried glassware using dry solvents and standard syringe
techniques. Tetrahydrofuran (THF) was distilled from the sodium
benzophenone ketyl radical ion. Dichloromethane was distilled
from calcium hydride. All reagents were of commercial quality
and used as received. After workup, partitioned organic layers
were washed with water and brine and dried over sodium sulfate
(Na2SO4). Reaction progress was monitored using aluminum-
backed thin layer chromatography (TLC) plates pre-coated with sil-
ica UV254. Purification of products by column chromatography
was conducted using a column filled with Silica Gel 60 (220–
240 mesh) using eluting solvent systems as indicated. Purification
by preparative thin layer chromatography (PTLC) was performed
using glass-backed plates pre-coated with silica UV254. HPLC
was conducted on Shimadzu SCL-10AVP system using a column
C
13H21NNaO6 m/z 310.1267.1341 ([M+Na]+), found m/z 310.1263.
5.1.4. (2S,4S)-N-Boc-4-(tert-butyldimethylsilyloxy)prolinemethyl
ester (11)
To a solution of 10 (9.7 g, 33.8 mmol) in methanol (250 mL) was
added K2CO3 (5.5 g, 36 mmol, 1.07 equiv). The mixture was stirred
for 1 h at rt and filtered. The solvent was evaporated in vacuo and
the residue purified by column chromatography (1:1 EtOAc–
hexanes) to yield (2S,4S)-N-Boc-4-hydroxy-proline methyl ester
as a colorless oil (6.5 g, 78%): ½a D23
ꢃ
ꢀ64.1 (c 0.5, MeOH); 1H NMR
purchased from Phenomenex (Luna 5
l
C18 (2) 25 ꢂ 4.6 mm). 1H
(500 MHz) d 4.29–4.24 (m, 7H), 3.75 (s, 1H), 3.77 (s, 2H), 3.66 (d,
J = 12.0 Hz, 0.5H), 3.59 (d, J = 12.0 Hz, 0.5H), 3.51 (m, 1.5 H), 3.29
(d, J = 9.5 Hz, 0.5H), 2.40–2.17 (m, 1H), 2.11–2.03 (m, 1H), 1.44 (s,
2.5H), 1.39 (s, 6.5H); 13C NMR (126 MHz) d 175.80, 175.50,
154.55, 153.77, 80.52, 71.36, 70.32, 60.48, 57.95, 57.76, 56.02,
55.43, 52.87, 52.58, 38.62, 37.79, 28.43, 28.32, 22.02, 21.12,
14.26; HRMS (ESI) calcd for C11H20NO5 m/z 246.1341 ([M+H]+),
found m/z 246.1325.
and 13C NMR spectra were recorded on a 400 MHz (400 MHz for
1H and 100 MHz for 13C) spectrometer or a 500 MHz (500 MHz
for 1H and 126 MHz for 13C) spectrometer in CDCl3. All chemical
shifts (d) were referenced to the solvent peaks of CDCl3
(7.26 ppm for 1H, 77.0 ppm for 13C). Optical rotation was measured
on a polarimeter operating at the sodium D-line.
5.1.2. (2S,4R)-N-Boc-4-hydroxy-proline methyl ester (9)
The product obtained above (6.5 g, 26.5 mmol), tert-butyldi-
methylsilyl chloride (9.6 g, 63.6 mmol, 2.4 equiv) and imidazole
(9.0 g, 132.5 mmol, 5 equiv) were stirred in DMF (50 mL) overnight
at rt. The reaction mixture was diluted with EtOAc (100 mL),
washed with water (10 mL ꢂ 2) and brine (10 mL ꢂ 2), dried with
anhydrous Na2SO4 and concentrated in vacuo. Purification by
column chromatography (1:4 EtOAc–hexanes) yielded 11 as a col-
A solution of trans-4-hydroxy-L-proline (5.24 g, 40 mmol) in
THF (50 mL) and water (25 mL) was treated with aqueous NaOH
(10%, 17 mL) and di-tert-butyl dicarbonate (13.1 g, 60 mmol,
1.5 equiv). The mixture was stirred overnight at room temperature.
THF was evaporated under reduced pressure. The aqueous solution
was adjusted to pH 2 with 10% aqueous NaHSO4 solution and ex-
tracted with EtOAc (3 ꢂ 50 mL). The combined organic phase was
washed, dried and concentrated to yield the crude product, which
was used in the next step without further purification.
To a solution of the above mentioned crude product in DMF
(180 mL) at room temperature was added Cs2CO3 (18.5 g,
56.8 mmol). After the mixture was stirred for 15 min, MeI
(4.05 mL, 59.4 mmol) was added dropwise. The resulting mixture
was then stirred overnight and filtered through a short column
orless oil (8.8 g, 93%). ½a D23
ꢃ
ꢀ35.0 (c 0.7, MeOH); 1H NMR (500 MHz)
d 4.43–4.21 (m, 2H), 3.67 (s, 3H), 3.60 (dd, J = 11.1, 5.4 Hz, 0.63H),
3.54 (dd, J = 11.1, 5.4 Hz, 1H), 3.30 (dd, J = 11.1, 3.6 Hz, 0.63 H), 3.25
(dd, J = 11.1, 3.2 Hz, 1H), 2.33–2.16 (m, 1H), 2.09–2.02 (m, 1H), 1.44
(s, 3H), 1.39 (s, 6H), 0.83 (s, 3H), 0.82 (s, 6H), 0.04–-0.02 (m, 6H);
13C NMR (126 MHz) d 172.95, 172.51, 154.44, 153.93, 79.96,
70.73, 69.80, 57.84, 57.44, 54.82, 54.25, 52.10, 51.96, 39.63,
38.81, 28.50, 28.38, 25.68, 25.66, 17.96, ꢀ4.88, ꢀ4.98; HRMS

J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
7671
(ESI) calcd for C17H34NO5Si m/z 360.2206 ([M+H]+), found m/z
360.2199.
2.58 (dd, J = 11.6, 4.3 Hz, 1H), 1.78–1.67 (m, 1H), 1.59–1.46 (m,
1H), 0.84 (s, 9H), ꢀ0.02 (s, 6H); 13C NMR (126 MHz) d 173.40,
129.80, 128.08, 127.89, 73.39, 58.25, 56.39, 41.37, 37.85, 25.84,
18.02, ꢀ4.70, ꢀ4.72; HRMS (ESI) calcd for C18H31N2O2Si m/z
335.2155 ([M+H]+), found m/z 335.2193.
5.1.5. (2S,4S)-tert-Butyl 4-(tert-butyldimethylsilyloxy)-2-(pheny
lcarbamoyl)pyrrolidine-1-carboxylate (13)
To a solution of 11 (8.8 g, 24.5 mmol) in methanol (45 mL) was
added aqueous lithium hydroxide (2.5 M, 15 mL). The mixture was
stirred for 6 h at 45 °C. The solution was adjusted to pH 3 with 3 M
HCl solution and extracted with EtOAc (30 mL ꢂ 3). The organic
solution was dried and concentrated at reduced pressure. Column
chromatography (5% MeOH in CH2Cl2) afforded the carboxylic acid
12 as a colorless oil (5.8 g, 77%).
5.1.8. 2-Iodo-3-methoxybenzaldehyde (16)
To a stirred solution of (2-iodo-3-methoxyphenyl)methanol
(15)29 (1.05 g, 4.0 mmol) in acetone (16 mL) at 0 °C was added
Jones’ reagent (1.5 mL) dropwise. The resulting yellow solution
was stirred at 0 °C for 10 min. Saturated aqueous NaHCO3
(10 mL) was added to quench the reaction. The mixture was ex-
tracted with ether (20 mL ꢂ 3) and the resulting organic solution
was dried and concentrated to afford crude 16 as a yellow solid
(0.9 g). This was used in the next step without further purification.
1H NMR (500 MHz) d 10.18 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.38 (t,
J = 7.8 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 3.94 (s, 3H); 13C NMR
(126 MHz) d 196.61, 158.37, 136.81, 129.58, 122.38, 116.11,
94.01, 56.93.
To a stirred solution of the acid (5.8 g, 16.8 mmol) and triethyl-
amine (2.6 mL, 18.5 mmol) in THF (60 mL) at 0 °C was added
methyl chloroformate (1.4 mL, 18.5 mmol, 1.1 equiv) dropwise.
After stirring for 1 h at 0 °C, aniline (1.7 mL, 18.5 mmol, 1.1 equiv)
was added. The resulting mixture was stirred for 1 h at 0 °C, 16 h at
rt and then 3 h under reflux. The reaction was cooled to rt and di-
luted with EtOAc (100 mL). The organic solution was washed, dried
and concentrated to produce the crude product, which was puri-
fied by column chromatography (1:6 EtOAc–hexanes) to yield
5.1.9. 3-Hydroxy-2-iodobenzaldehyde (17)
To a cooled solution (0 °C) of 16 (0.86 g, 3.3 mmol) in CH2Cl2
(30 mL) was added a 1 M solution of BBr3 in CH2Cl2 (8.2 mL,
8.2 mmol, 2.5 equiv) dropwise. The resulting solution was brought
to rt and stirred for 4 h. The reaction was stopped by adding cold
water (10 mL). The aqueous phase was extracted with CH2Cl2
(20 mL ꢂ 3) and the combined organic phase was washed, dried
and concentrated. The crude product was purified by column chro-
matography (1:4 EtOAc–hexanes) to give compound 17 as a crys-
talline solid (0.40 g, 49%). 1H NMR (500 MHz) d 10.03 (s, 1H),
7.45 (dd, J = 7.5, 1.6 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.26 (dd,
J = 8.1, 1.5 Hz, 1H); 13C NMR (126 MHz) d 194.69, 155.56, 135.79,
130.01, 124.13, 120.83, 100.00.
compound 13 as a colorless oil (5.5 g, 78%). ½a D23
ꢀ32.8 (c 0.7,
ꢃ
MeOH); 1H NMR (500 MHz) d 7.50 (d, J = 7.9 Hz, 2H), 7.29 (t,
J = 7.6 Hz, 2H), 7.07 (t, J = 6.9 Hz, 1H), 4.38 (m, 2H), 3.70–3.26 (br
s, 2H), 2.27 (br s, 2H), 1.42 (br s, 9H), 0.76 (br s, 9H); 13C NMR
(126 MHz) d 170.99, 155.15, 128.96, 124.07, 119.52, 81.24, 70.70,
61.41, 56.39, 39.65, 28.40, 25.67, 18.10, ꢀ4.85; HRMS (ESI) calcd
for C22H37N2O4Si m/z 421.2523 ([M+H]+), found m/z 421.2516.
5.1.6. (2S,4S)-tert-Butyl 4-(tert-butyldimethylsilyloxy)-2-(N-
methyl-N-phenylcarbamoyl)-pyrrolidine-1-carboxylate (14)
To a solution of 13 (5.4 g, 12.8 mmol) in DMF (75 mL) was
added NaH (3.1 g, 60%, dispersed in mineral oil, 77.5 mmol,
6.6 equiv) at 0 °C and the resulting mixture was stirred at the same
temperature for 1 h, followed by the slow addition of MeI (4.8 mL,
76.6 mmol, 6 equiv). The reaction was stirred for another 15 min at
0 °C and quenched by adding cold water. The aqueous phase was
extracted with EtOAc (50 mL ꢂ 3) and the combined organic phase
was washed, dried and concentrated. The crude product was puri-
fied with column chromatography (1:4 EtOAc–hexanes) to afford
5.1.10. 3-(Allyloxy)-2-iodobenzaldehyde (8)
A solution of 17 (0.38 g, 1.5 mmol) in DMF (10 mL) was treated
with K2CO3 (0.63 g, 4.5 mmol, 3 equiv) and allybromide (0.15 mL,
1.7 mmol, 1.1 equiv). The resulting suspension was stirred for 6 h
at rt. Water (5 mL) and ether (15 mL) were added to the reaction
mixture and the aqueous phase was extracted with ether
(15 mL ꢂ 2). The combined ether layers was dried and concen-
trated. The residue was purified by column chromatography (1:9
EtOAc–hexanes) to afford compound 8 as a colorless oil (0.42 g,
96%). 1H NMR (400 MHz) d 10.20 (s, 1H), 7.50 (d, J = 8.6 Hz, 1H),
7.36 (t, J = 7.9 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 6.16–6.00 (m, 1H),
5.56 (d, J = 17.2 Hz, 1H), 5.36 (d, J = 10.6 Hz, 1H), 4.65 (s, 2H); 13C
NMR (126 MHz) d 196.66, 157.39, 136.88, 132.13, 129.47, 122.58,
118.21, 117.65, 94.67, 70.32.
14 as a crystalline solid (5.2 g, 93%). ½a D23
ꢀ22.1 (c 1.0, MeOH);
ꢃ
1H NMR (500 MHz) d 7.52–7.28 (m, 5H), 7.21 (d, J = 8.5 Hz, 1H),
4.22–3.99 (m, 2H), 3.78–3.58 (m, 1H), 3.26 (s, 3H), 3.23–3.16 (m,
1H), 2.06–1.91 (m, 1H), 1.89–1.69 (m, 1H); 13C NMR (126 MHz) d
171.88, 154.16, 153.59, 143.50, 143.19, 129.86, 129.76, 128.62,
128.17, 127.99, 127.85, 127.75, 125.72, 124.81, 79.89, 79.51,
69.55, 68.91, 55.66, 55.30, 53.38, 52.81, 39.52, 38.52, 37.88,
37.82, 28.59, 28.54, 25.69, 17.91, 17.89, ꢀ4.82, ꢀ4.83, ꢀ4.86,
ꢀ4.95; HRMS (ESI) calcd for
C23H39N2O4Si m/z 435.2679
5.1.11. (2S,4S)-1-(3-(Allyloxy)-2-iodobenzyl)-4-(tert-butyldi
methylsilyloxy)-N-methyl-N-phenyl-pyrrolidine-2-carbox
amide (18)
([M+H]+), found m/z 435.2714.
5.1.7. (2S,4S)-4-(tert-Butyldimethylsilyloxy)-N-methyl-N-phenyl
pyrrolidine-2-carboxamide (7)
A mixture of 8 (0.60 g, 2.1 mmol) and 7 (0.70 g, 2.1 mmol,
1 equiv) in 1,2-dichloroethane (15 mL) was treated with NaB-
H(OAc)3 (0.62 g, 2.9 mmol, 1.4 equiv). The resulting solution was
stirred at rt overnight followed by the addition of saturated aque-
ous NaHCO3 (5 mL). The aqueous phase was extracted with CH2Cl2
(15 mL ꢂ 2) and the combined organic phase was dried and con-
centrated in vacuo. The residue was purified by column chroma-
tography (11% isopropanol in hexanes) to afford 18 as a colorless
To a cold solution (0 °C) of 14 (0.94 g, 2.2 mmol) in CH2Cl2
(100 mL) was added ZnBr2 (2.43 g, 10.8 mmol, 4.9 equiv) and the
resulting suspension was warmed up to rt and stirred overnight.
The reaction was quenched by adding saturated aqueous NaHCO3
(20 mL). The aqueous phase was extracted with CH2Cl2
(40 mL ꢂ 3) and the combined organic phase was dried and con-
centrated. The crude product was purified by column chromatog-
raphy (50% EtOAc in hexanes, then 10% methanol in CH2Cl2) to
oil (1.1 g, 87%). ½a D23
ꢃ
ꢀ49.4 (c 0.5, MeOH); 1H NMR (400 MHz) d
7.34 (m, 3H), 7.21 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 6.8 Hz, 1H), 7.01
(d, J = 7.2 Hz, 1H), 6.71 (d, J = 8.5 Hz, 1H), 6.17–6.02 (m, 1H), 5.57
(ddd, J = 17.2, 3.2, 1.4 Hz, 1H), 5.33 (ddd, J = 10.4 Hz, 4.1, 1.5 Hz,
1H), 4.62 (dt, J = 4.7, 1.6 Hz, 1H), 4.32–4.18 (m, 1H), 3.88 (d,
J = 14.5 Hz, 1H), 3.73 (d, J = 14.8 Hz, 1H), 3.45 (t, J = 7.1 Hz, 1H),
yield compound 7 as a colorless oil (0.69 g, 93%). ½a D23
ꢀ46.4 (c
ꢃ
0.5, CHCl3); 1H NMR (500 MHz) d 7.40 (t, J = 7.5 Hz, 2H), 7.33 (t,
J = 7.5 Hz, 1H), 7.18 (d, J = 7.5 Hz, 2H), 4.14 (br, 1H), 3.48 (t,
J = 8.1 Hz, 1H), 3.27 (s, 3H), 2.92 (d, J = 11.6 Hz, 1H), 2.74 (s, 2H),

7672
J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
3.27 (s, 3H), 3.17 (dd, J = 9.0, 6.0 Hz, 1H), 2.73–2.65 (m, 1H), 2.18–
2.08 (m, 1H), 2.00–1.89 (m, 1H), 0.87 (s, 9H), 0.01 (s, 6H); 13C NMR
(126 MHz) d 173.26, 156.98, 143.57, 132.84, 129.77, 129.34,
128.81, 127.83, 127.57, 123.07, 121.14, 118.19, 117.73, 117.59,
111.64, 111.00, 92.64, 70.63, 70.02, 69.78, 61.31, 60.93, 60.28,
40.06, 37.79, 25.91, 18.12, 1.10, 0.08, ꢀ4.66, ꢀ4.67, ꢀ4.69, ꢀ4.70;
HRMS (ESI) calcd for C28H40IN2O3Si m/z 607.1853 ([M+H]+), found
m/z 607.1883.
109.52, 100.87, 69.98, 68.93, 65.09, 63.58, 63.17, 55.05, 36.59;
HRMS (ESI) calcd for C22H24NO4 m/z 366.1705 ([M+H]+), found
m/z 366.1729.
5.1.14. (2S,10R,10aS)-9-Allyloxy-2,10-dihydroxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinoline (22) and (2S,10S,10aS)-9-
allyloxy-2,10-dihydroxy-1,2,3,5,10,10a-hexahydropyrrolo[1,2-
b]isoquinoline (23)
A solution of 18 (470 mg, 0.77 mmol) in THF (15 mL) was cooled
to ꢀ78 °C and a 2.5 M solution of n-BuLi in hexanes (0.34 mL,
0.85 mmol) was added over 10 min. The resulting yellow solution
was stirred for 3 h at ꢀ78 °C and quenched by adding saturated
aqueous NaHCO3 (8 mL). The aqueous phase was extracted with
EtOAc (20 mL ꢂ 2). The combined organic layers were dried and
concentrated to afford the crude ketone. A solution of this ketone
in THF (8 mL) was cooled to 0 °C and treated with HF (0.3 mL,
70% in pyridine). The reaction mixture was warmed up to rt and
stirred overnight. Saturated aqueous NaHCO3 (15 mL) was added
carefully to quench the unreacted HF and the resulting solution
was extracted with EtOAc (20 mL ꢂ 3). The combined organic solu-
tion was washed, dried and concentrated to afford the crude prod-
uct containing the deprotected ketone as an orange oil. To a stirred
solution of this crude product in methanol (5 mL) was added
NaBH4 (117 mg, 3.1 mmol) in portions. After stirring for 1 h at rt
saturated aqueous NaHCO3 (15 mL) was added slowly and the
resulting solution was extracted with diethyl ether (20 mL ꢂ 3).
The combined ether layers were dried and concentrated. The
resulting crude product was purified by a short silica gel column
and eluted first with hexanes–EtOAc = 1: 2 (50 mL) and then
CH2Cl2–MeOH = 9: 1 (50 mL). The second eluate was concentrated
to give a diastereomeric mixture of 22 and 23. The mixture was
further separated by a short column packed with reversed phase
silica gel C-18 (40% methanol in water) to afford 22 (59 mg, 29%
for 3 steps) and 23 (40 mg, 20% for 3 steps) as white crystalline sol-
5.1.12. (2S,10S,10aS)-9-Allyloxy-10-hydroxy-2-(tert-butyldimeth
ylsilyloxy)-1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinoline
(20)
A solution of 18 (150 mg, 0.25 mmol) in THF (10 mL) was cooled
to ꢀ78 °C and a 2.5 M solution of n-BuLi in hexanes (0.1 mL,
0.25 mmol) was added over 10 min. The resulting yellow solution
was stirred for 3 h at ꢀ78 °C and quenched by adding saturated
aqueous NaHCO3 (5 mL). The aqueous phase was extracted with
EtOAc (10 mL ꢂ 2) and the combined organic phase was dried
and concentrated to afford the crude ketone 19. This was used in
the next step without further purification.
Crude ketone 19 from the previous step was dissolved in MeOH
(5 mL) and the resulting solution was treated with NaBH4 (38 mg,
1 mmol). After stirring for 1 h at rt, saturated aqueous NaHCO3
(5 mL) was added carefully to quench the unreacted hydride. To
the resulting solution was added ether (20 mL). The aqueous phase
was extracted with ether (20 mL ꢂ 2) and the combined organic
phase was washed, dried and concentrated. The residue was
purified by preparative thin layer chromatography (PTLC) (1:3
EtOAc–hexanes) to yield 20 as a colorless oil (53 mg, 57% for two
steps). ½a 2D3
ꢃ
+13.7 (c 1.0, MeOH); 1H NMR (500 MHz) d 7.16 (t,
J = 7.9 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 6.67 (d, J = 7.7 Hz, 1H),
6.08 (ddt, J = 17.2, 10.4, 5.1 Hz, 1H), 5.43 (dq, J = 17.3, 1.6 Hz, 1H),
5.26 (dq, J = 10.5, 1.4 Hz, 1H), 4.86 (dd, J = 9.7, 1.7 Hz, 1H), 4.66
(ddt, J = 12.9, 5.0, 1.6 Hz, 1H), 4.55 (ddt, J = 13.0, 5.3, 1.5 Hz, 1H),
4.45–4.38 (m, 1H), 4.10 (d, J = 14.9 Hz, 1H), 3.32 (d, J = 14.8 Hz,
1H), 3.16 (d, J = 9.7 Hz, 1H), 2.62 (d, J = 9.9 Hz, 1H), 2.48–2.40 (m,
2H), 2.26–2.15 (m, 2H), 0.88 (s, 9H), 0.07 (s, 3H), 0.07 (s, 3H); 13C
NMR (126 MHz) d 157.14, 136.70, 133.48, 128.40, 127.18, 118.89,
117.44, 109.68, 70.55, 69.06, 64.84, 64.00, 61.87, 55.62, 36.49,
26.02, 18.26, ꢀ4.52, ꢀ4.70; HRMS (ESI) calcd for C21H34NO3Si m/z
376.2364 ([M+H]+), found m/z 376.2308.
ids. Compound 22: ½a D23
ꢃ
+100.3 (c 0.3, CHCl3); 1H NMR (500 MHz) d
7.15 (t, J = 7.9 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 6.72 (d, J = 7.7 Hz,
1H), 6.06 (qd, J = 10.6, 5.3 Hz, 1H), 5.41 (d, J = 17.3 Hz, 1H), 5.32
(d, J = 10.5 Hz, 1H), 4.90 (d, J = 8.7 Hz, 1H), 4.61 (d, J = 5.1 Hz, 2H),
4.36 (t, J = 4.7 Hz, 1H), 4.20 (s, 1H), 3.97 (d, J = 14.4 Hz, 1H), 3.39
(d, J = 14.4 Hz, 1H), 3.13 (d, J = 10.0 Hz, 1H), 2.79 (dt, J = 14.9,
7.6 Hz, 1H), 2.41 (dd, J = 10.0, 4.9 Hz, 1H), 2.30 (q, J = 8.3 Hz, 1H),
1.76 (dd, J = 13.5, 8.5 Hz, 1H); 13C NMR (126 MHz) d 157.18,
137.07, 132.59, 128.13, 126.73, 119.56, 118.64, 110.00, 72.45,
70.35, 69.20, 65.85, 63.89, 55.54, 41.60; HRMS (ESI) calcd for
5.1.13. (2S,10S,10aS)-9-Allyloxy-10-benzoyloxy-2-hydroxy-
1,2,3,5,10,10a-hexahydropyrrolo[1,2-b]isoquinoline (21)
To a cooled (0 °C) solution of 20 (55 mg, 0.15 mmol) in THF
(6 mL) was added slowly a 1.0 M solution of LiHMDS in THF
(0.22 mL, 0.22 mmol, 1.5 equiv) and the resulting solution was stir-
red for 10 min at 0 °C. Benzoyl chloride (0.034 mL, 0.30 mmol,
2 equiv) was added to the mixture in one portion and the reaction
was stirred for 4 h at the same temperature. Saturated aqueous
NaHCO3 (5 mL) was added. Usual workup afforded the crude prod-
uct as a brown gum. The above crude product was dissolved in
5 mL THF and cooled to 0 °C. To this solution was added HF
(0.15 mL, 70% in pyridine) and warmed up to rt overnight. Satu-
rated aqueous NaHCO3 (10 mL) was added slowly and the resulting
solution was extracted with EtOAc (20 mL ꢂ 3). The EtOAc solution
was washed, dried and concentrated. The crude product was puri-
fied by PTLC (3:1 EtOAc–hexanes) to yield compound 21 (32 mg,
C
15H20NO3 m/z 262.1443 ([M+H]+), found m/z 262.1423. 23: ½a 2D3
ꢃ
+63.8 (c 0.6, CHCl3); 1H NMR (500 MHz) d 7.18 (t, J = 7.9 Hz, 1H),
6.75 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 7.7 Hz, 1H), 6.09 (ddt, J = 17.2,
10.2, 5.1 Hz, 1H), 5.46 (dd, J = 17.3, 1.5 Hz, 1H), 5.29 (dd, J = 10.6,
1.4 Hz, 1H), 4.82 (s, 1H), 4.64 (dd, J = 12.9, 4.8 Hz, 1H), 4.56 (dd,
J = 12.9, 5.3 Hz, 1H), 4.28 (t, J = 6.5 Hz, 1H), 3.37 (d, J = 14.9 Hz,
1H), 3.06 (d, J = 14.8 Hz, 1H), 2.95 (d, J = 10.1 Hz, 1H), 2.37–2.22
(m, 3H), 2.22–2.16 (m, 1H); 13C NMR (126 MHz)
d 157.25,
136.35, 133.26, 128.49, 126.34, 119.24, 117.57, 109.53, 77.36,
77.11, 76.86, 69.93, 68.98, 65.12, 64.18, 61.80, 54.84, 36.04; HRMS
(ESI) calcd for C15H20NO3 m/z 262.1443 ([M+H]+), found m/z
262.1424.
5.1.15. (2S,10S,10aS)-9-Allyloxy-10-benzoyloxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-30-benzoyla
mino-20-hydroxy-30-(2-(allyloxy)phenyl)-propanoate (28)
To a stirred suspension of NaH (100 mg, 60%, dispersed in min-
eral oil, 2.5 mmol, 25 equiv) in THF (1 mL) at 0 °C was added a solu-
tion of 21 (36.5 mg, 0.1 mmol, 1 equiv) in THF (1 mL) and the
resulting mixture was stirred for 10 min at 0 °C. To this mixture
was added a solution of 2532a (75 mg, 0.16 mmol, 1.6 equiv) in
THF (1 mL). The reaction was allowed to warm up gradually to rt
59% for two steps) as a white solid. ½a D23
ꢃ
+50.9 (c 0.4, MeOH); 1H
NMR (400 MHz) d 8.01 (m, 2H), 7.49 (m, 1H), 7.41–7.33 (m, 2H),
7.26 (m, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.72 (d, J = 8.2 Hz, 1H), 6.54
(d, J = 2.4 Hz, 1H), 5.87–5.72 (m, 1H), 5.20 (m, 1H), 5.08 (m, 1H),
4.49–4.36 (m, 2H), 4.23 (m, 2H), 3.40 (d, J = 14.8 Hz, 1H), 3.21 (d,
J = 9.8 Hz, 1H), 2.62 (t, J = 8.4 Hz, 1H), 2.49–2.34 (m, 2H), 1.81–
1.66 (m, 1H); 13C NMR (126 MHz) d 166.48, 157.71, 138.01,
132.78, 130.56, 129.91, 129.41, 128.27, 122.03, 118.82, 117.38,

J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
7673
overnight and quenched by careful addition of excess aqueous
NaHCO3. The aqueous phase was extracted with EtOAc
(10 mL ꢂ 3) and the combined organic phase was dried and con-
centrated. Purification by PTLC (hexanes–EtOAc = 2:1) yielded a
(m, 2H), 2.45–2.27 (m, 2H), 1.96–1.86 (m, 1H), 1.01–0.60 (m,
21H); 13C NMR (125 MHz) d 172.53, 166.73, 166.37, 157.54,
156.00, 137.69, 134.62, 134.58, 132.70, 131.38, 130.68, 130.06,
129.41, 128.85, 128.52, 128.25, 127.91, 127.30, 126.39, 121.56,
120.42, 118.81, 117.37, 117.35, 111.07, 109.38, 73.59, 72.89,
68.89, 67.17, 64.88, 63.55, 61.27, 55.13, 53.92, 33.52, 32.43,
17.71, 17.66, 12.16; HRMS (ESI) calcd for C51H63N2O8Si m/z
859.4354 ([M+H]+), found m/z 859.4358.
colorless oil (82 mg, 97%). ½a D23
ꢃ
ꢀ16.6 (c 1.0, MeOH); 1H NMR
(500 MHz) d 8.06 (dd, J = 8.3, 1.3 Hz, 2H), 7.88–7.83 (m, 2H), 7.50
(t, J = 7.4 Hz, 1H), 7.39–7.33 (m, 3H), 7.32–7.21 (m, 4H), 7.17–
7.12 (m, 1H), 7.09 (d, J = 7.8 Hz, 1H), 6.82 (t, J = 7.5 Hz, 1H), 6.74–
6.67 (m, 2H), 6.64 (d, J = 7.8 Hz, 1H), 6.49 (d, J = 3.2 Hz, 1H),
5.88–5.68 (m, 3H), 5.37 (dd, J = 17.3, 1.5 Hz, 1H), 5.25–5.20 (m,
1H), 5.18 (ddd, J = 17.3, 3.1, 1.6 Hz, 1H), 5.09–5.03 (m, 2H), 4.87
(d, J = 2.7 Hz, 1H), 4.41 (qd, J = 12.7, 5.2 Hz, 2H), 4.32 (dt, J = 4.7,
1.3 Hz, 2H), 3.87 (d, J = 15.0 Hz, 1H), 3.23–3.14 (m, 2H), 2.62–2.48
(m, 2H), 2.45–2.36 (m, 1H), 1.95–1.86 (m, 1H), 0.93–0.82 (m,
3H), 0.80–0.69 (m, 18H); 13C NMR (125 MHz) d 172.55, 166.61,
166.38, 157.56, 155.71, 137.64, 134.58, 132.71, 132.70, 131.39,
130.67, 130.08, 129.43, 128.82, 128.55, 128.26, 127.98, 127.24,
121.54, 120.64, 118.81, 117.38, 111.65, 109.41, 77.29, 73.54,
72.96, 68.90, 68.59, 64.89, 63.59, 61.31, 55.19, 53.80, 32.44,
17.71, 17.67, 12.16; HRMS (ESI) calcd for C50H61N2O8Si m/z
845.4197 ([M+H]+), found m/z 845.4203.
A solution of the above product (8 mg, 0.009 mmol) in THF
(3 mL) was cooled to 0 °C with an ice bath and treated with HF
(0.1 mL, 70% in pyridine). The ice bath was removed and the reac-
tion was continued overnight. Saturated aqueous NaHCO3 (10 mL)
was added slowly and the resulting solution was extracted with
EtOAc (20 mL ꢂ 3). The EtOAc solution was washed, dried and con-
centrated. The crude product was purified by PTLC (1:1 EtOAc–
hexanes) to yield compound 27 (5.5 mg, 85%) as an amorphous so-
lid. ½a 2D3
ꢃ
ꢀ31.8 (c 0.5, MeOH); 1H NMR (500 MHz) d 8.10–8.05 (m,
2H), 7.75–7.67 (m, 2H), 7.54–7.48 (m, 1H), 7.48–7.42 (m, 1H),
7.41–7.33 (m, 5H), 7.24–7.14 (m, 2H), 7.07 (dd, J = 7.6, 1.6 Hz,
1H), 6.84–6.79 (m, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.70 (d, J = 8.3 Hz,
1H), 6.52 (d, J = 8.2 Hz, 1H), 6.42 (d, J = 3.0 Hz, 1H), 5.82–5.60 (m,
2H), 5.47 (dd, J = 9.1, 6.9 Hz, 1H), 5.15 (dd, J = 17.3, 1.5 Hz, 1H),
5.07–4.96 (m, 3H), 4.90 (dd, J = 10.2, 1.2 Hz, 1H), 4.50 (m, 1H),
4.44–4.29 (m, 2H), 4.11 (m, 2H), 3.72 (ddd, J = 9.0, 7.4, 5.8 Hz,
1H), 3.52–3.32 (m, 2H), 3.29 (d, J = 11.5 Hz, 1H), 3.24 (d,
J = 14.9 Hz, 1H), 2.53–2.38 (m, 2H), 2.32–2.18 (m, 2H), 2.17–2.05
(m, 1H); 13C NMR (126 MHz) d 173.08, 167.91, 166.23, 157.53,
156.58, 137.39, 134.47, 134.35, 132.85, 132.64, 131.59, 130.62,
130.18, 129.80, 129.54, 129.46, 128.48, 128.31, 127.29, 124.94,
121.37, 121.04, 118.79, 117.88, 117.39, 111.62, 109.48, 74.03,
73.01, 68.87, 66.77, 64.80, 63.78, 61.21, 56.39, 55.34, 33.68,
32.36, 29.78; HRMS (ESI) calcd for C42H43N2O8 m/z 703.3014
([M+H]+), found m/z 703.3027.
A
solution of the compound obtained above (18 mg,
0.038 mmol) in THF (5 mL) was cooled to 0 °C with an ice bath
and treated with HF pyridine (0.15 mL, 70%). The ice bath was re-
moved and the reaction was continued overnight. Saturated aque-
ous NaHCO3 (10 mL) was added slowly and the resulting solution
was extracted with EtOAc (20 mL ꢂ 3). The EtOAc solution was
washed, dried and concentrated. The crude product was purified
by PTLC (50% EtOAc in hexanes) to yield compound 28 (11.5 mg,
84%) as a white solid. ½a D23
ꢃ
ꢀ47.8 (c 0.5, MeOH); 1H NMR
(400 MHz) d 8.08 (d, J = 7.8 Hz, 2H), 7.73 (d, J = 7.8 Hz, 2H), 7.52
(t, J = 7.4 Hz, 1H), 7.49–7.32 (m, 6H), 7.25–7.16 (m, 2H), 7.08 (d,
J = 7.4 Hz, 1H), 6.84 (t, J = 7.5 Hz, 1H), 6.73 (d, J = 7.8 Hz, 1H), 6.70
(d, J = 8.2 Hz, 1H), 6.52 (d, J = 8.3 Hz, 1H), 6.43 (d, J = 2.9 Hz, 1H),
5.85–5.67 (m, 2H), 5.52 (dd, J = 9.0, 6.8 Hz, 1H), 5.28 (m, 2H),
5.20–5.12 (m, 2H), 5.05 (d, J = 10.4 Hz, 2H), 4.52 (br s, 1H), 4.38
(ddd, J = 35.2, 12.6, 5.1 Hz, 2H), 4.19–4.08 (m, 2H), 3.99 (dd,
J = 12.6, 4.8 Hz, 1H), 3.33 (d, J = 11.5 Hz, 1H), 3.26 (d, J = 15.1 Hz,
1H), 2.56–2.40 (m, 2H), 2.23–2.10 (m, 1H); 13C NMR (126 MHz) d
173.07, 167.39, 166.28, 157.54, 156.19, 137.37, 134.30, 132.84,
132.65, 132.52, 131.60, 130.63, 130.20, 129.68, 129.54, 129.31,
128.53, 128.30, 127.14, 125.22, 121.40, 121.19, 118.78, 118.18,
117.39, 112.06, 109.49, 73.87, 73.20, 68.89, 68.64, 64.81, 63.79,
61.22, 55.78, 55.32, 32.38; HRMS (ESI) calcd for C41H41N2O8 m/z
689.2863 ([M+H]+), found m/z 689.2858.
5.1.17. (2S,10S,10aS)-9-Allyloxy-10-benzoyloxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-30-benzoyla
mino-20-hydroxy-30-(2-allylphenyl)-propanoate (29)
To a stirred suspension of NaH (41 mg, 60%, dispersed in min-
eral oil, 1.0 mmol, 25 equiv) in THF (1.5 mL) at 0 °C was added
slowly a solution of 21 (15 mg, 0.041 mmol) in THF (1.5 mL) and
the resulting mixture was stirred for 10 min at 0 °C. To this mixture
was added a solution of 3.3732a (28.5 mg, 0.062 mmol, 1.5 equiv) in
THF (1.5 mL). The reaction was allowed to warm up gradually to rt
overnight and quenched by careful addition of excess aqueous
NaHCO3. The aqueous phase was extracted with EtOAc
(10 mL ꢂ 3) and the combined organic phase was dried and con-
centrated. Purification by PTLC (hexanes–EtOAc = 2:1) yielded a
5.1.16. (2S,10S,10aS)-9-Allyloxy-10-benzoyloxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-30-benzoyla
mino-20-hydroxy-30-(2-(but-3-enyloxy)phenyl)-propanoate (27)
To a stirred suspension of NaH (79 mg, 60%, dispersed in min-
eral oil, 2.0 mmol, 27 equiv) in THF (2.5 mL) at 0 °C was added
slowly a solution of 21 (27 mg, 0.074 mmol) in THF (2.5 mL) and
the resulting mixture was stirred for 10 min at 0 °C. To this mixture
was added a solution of 2432a (61 mg, 0.12 mmol, 1.7 equiv) in THF
(2.5 mL). The reaction was allowed to warm up gradually to rt
overnight and quenched by careful addition of excess aqueous
NaHCO3. The aqueous phase was extracted with EtOAc
(10 mL ꢂ 3) and the combined organic phase was dried and con-
centrated. Purification by PTLC (hexanes–EtOAc = 2:1) yielded a
colorless oil (28 mg, 83%). ½a D23
ꢃ
ꢀ24.8 (c 0.8, CHCl3); 1H NMR
(500 MHz) d 8.04 (d, J = 7.5 Hz, 2H), 7.84 (d, J = 7.4 Hz, 2H), 7.51
(t, J = 7.4 Hz, 1H), 7.36 (m, 3H), 7.28 (m, 3H), 7.25–7.22 (m, 1H),
7.20–7.10 (m, 2H), 7.10–7.02 (m, 2H), 6.73 (d, J = 8.2 Hz, 1H),
6.64 (d, J = 7.7 Hz, 1H), 6.52 (d, J = 3.0 Hz, 1H), 5.82–5.71 (m, 2H),
5.62 (t, J = 8.1 Hz, 1H), 5.32–5.25 (m, 2H), 5.18 (d, J = 17.3 Hz,
1H), 5.06 (d, J = 9.8 Hz, 1H), 4.54 (s, 1H), 4.42 (qd, J = 12.7, 5.2 Hz,
2H), 3.90 (d, J = 15.0 Hz, 1H), 3.40 (dd, J = 15.9, 5.6 Hz, 1H), 3.30–
3.11 (m, 3H), 2.65–2.44 (m, 3H), 2.10–1.96 (m, 1H), 0.83–0.63
(m, 21H); 13C NMR (126 MHz) d 172.37, 166.60, 166.45, 137.55,
137.02, 136.97, 136.28, 136.26, 132.75, 132.69, 131.50, 131.49,
130.56, 130.30, 130.07, 129.45, 128.58, 128.24, 127.83, 127.24,
126.89, 126.22, 121.48, 118.81, 117.40, 116.49, 109.45, 74.65,
73.48, 68.91, 64.81, 63.77, 61.52, 55.14, 53.63, 36.53, 32.50,
29.78, 17.70, 17.66, 12.20; HRMS (ESI) calcd for C50H61N2O7Si m/z
829.4248 ([M+H]+), found m/z 829.4196.
colorless oil (44 mg, 72%). ½a D23
ꢃ
ꢀ27.4 (c 0.5, MeOH); 1H NMR
(500 MHz) d 8.05 (d, J = 7.8 Hz, 2H), 7.86 (d, J = 7.7 Hz, 2H), 7.50
(t, J = 7.4 Hz, 1H), 7.43–7.13 (m, 8H), 7.08 (d, J = 7.4 Hz, 1H), 6.80
(t, J = 7.5 Hz, 1H), 6.71 (t, J = 7.6 Hz, 2H), 6.62 (d, J = 7.7 Hz, 1H),
6.49 (d, J = 3.0 Hz, 1H), 5.86–5.67 (m, 3H), 5.29–4.94 (m, 5H),
4.88 (d, J = 2.6 Hz, 1H), 4.41 (ddd, J = 27.5, 12.6, 5.0 Hz, 2H), 3.85
(ddd, J = 23.5, 17.9, 12.2 Hz, 3H), 3.22–3.11 (m, 2H), 2.63–2.46
A solution of the above compound (5 mg, 0.006 mmol) in THF
(3 mL) was cooled to 0 °C with an ice bath and treated with HF
(0.1 mL, 70% in pyridine). The ice bath was removed and the reac-

7674
J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
tion was continued overnight. Saturated aqueous NaHCO3 (10 mL)
was added slowly and the resulting solution was extracted with
EtOAc (10 mL ꢂ 3). The EtOAc solution was washed, dried and
concentrated. The crude product was purified by PTLC (1:1
EtOAc–hexanes) to yield compound 29 (3.5 mg, 87%) as a colorless
CHCl3); 1H NMR (500 MHz) d 7.87–7.83 (m, 2H), 7.39 (dt, J = 2.4,
1.8 Hz, 1H), 7.34 (dd, J = 8.1, 6.4 Hz, 3H), 7.19 (dt, J = 16.0, 4.8 Hz,
3H), 6.89–6.83 (m, 2H), 6.75 (d, J = 8.1 Hz, 1H), 6.62 (d, J = 7.7 Hz,
1H), 6.18–6.03 (m, 2H), 5.89–5.82 (m, 1H), 5.57–5.51 (m, 1H),
5.48–5.40 (m, 1H), 5.35–5.20 (m, 4H), 4.98 (d, J = 2.0 Hz, 1H),
4.89–4.83 (m, 1H), 4.68 (ddt, J = 13.1, 4.9, 1.5 Hz, 1H), 4.63–4.53
(m, 3H), 3.86 (d, J = 15.0 Hz, 1H), 3.25–3.15 (m, 2H), 2.53 (dd,
J = 11.3, 6.1 Hz, 1H), 2.48–2.36 (m, 2H), 2.33–2.26 (m, 1H), 2.21
(d, J = J = 9.8 Hz, 1H), 0.95–0.81 (m, 21H); 13C NMR (126 MHz) d
172.48, 166.35, 157.09, 155.72, 136.13, 134.56, 133.40, 133.09,
131.50, 128.79, 128.65, 128.56, 127.74, 127.06, 126.86, 126.63,
120.62, 118.93, 117.55, 117.53, 111.66, 109.83, 73.83, 73.57,
69.10, 68.92, 64.57, 61.51, 61.50, 55.40, 53.07, 31.68, 17.82,
17.81, 17.76, 12.31; HRMS (ESI) calcd for C43H57N2O7Si m/z
741.3935 ([M+H]+), found m/z 741.3915.
oil. ½a 2D3
ꢃ
ꢀ16.0 (c 0.3, CHCl3); 1H NMR (500 MHz) d 8.00 (d,
J = 7.9 Hz, 2H), 7.71 (d, J = 7.8 Hz, 2H), 7.47–7.42 (m, 1H), 7.39
(m, 2H), 7.34 (m, 2H), 7.31–7.22 (m, 3H), 7.18 (t, J = 7.4 Hz, 1H),
7.12–7.05 (m, 2H), 6.76–6.68 (m, 3H), 6.54 (d, J = 2.8 Hz, 1H),
5.83–5.64 (m, 3H), 5.27 (t, J = 8.6 Hz, 1H), 5.18 (d, J = 17.3 Hz,
1H), 5.06 (d, J = 10.6 Hz, 1H), 4.85–4.77 (m, 2H), 4.48–4.32 (m,
3H), 4.15 (d, J = 14.9 Hz, 1H), 3.43 (d, J = 11.6 Hz, 1H), 3.31 (d,
J = 14.8 Hz, 1H), 3.26 (m, 2H), 2.68–2.47 (m, 3H), 1.95 (m, 1H).13
C
NMR (126 MHz) d 173.09, 166.84, 166.49, 157.60, 138.03, 137.36,
137.08, 136.47, 134.14, 132.77, 132.69, 131.68, 130.47, 130.43,
130.02, 129.54, 128.61, 128.18, 128.16, 127.16, 126.89, 126.76,
121.55, 118.78, 117.45, 116.33, 109.54, 77.35, 77.09, 76.84, 74.78,
72.90, 68.93, 64.70, 63.94, 61.01, 55.28, 51.55, 36.91, 29.78; HRMS
(ESI) calcd for C41H41N2O7 m/z 673.2914 ([M+H]+), found m/z
673.2878.
5.1.20. (2S,10S,10aS)-9-Allyloxy-10-hydroxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-30-benzoyla
mino-20-triisopropylsilyloxy-30-(2-allylphenyl)-propanoate (32)
A solution of 23 (18 mg, 0.069 mmol) in THF (1.5 mL) was added
into a stirred cold (0 °C) suspension of NaH (66 mg, 60%, dispersed
in mineral oil, 1.65 mmol, 24 equiv) in THF (1.5 mL) and the result-
ing mixture was stirred for 10 min at 0 °C. To this mixture was
added a solution of 26 (33.6 mg, 0.072 mmol, 1.05 equiv) in THF
(1.5 mL) and the reaction was allowed to continue for 20 min at
0 °C. Aqueous NaHCO3 (5 mL) was added into the reaction mixture
and the aqueous phase was extracted with EtOAc (10 mL ꢂ 3) and
the combined organic phase was dried and concentrated. Purifica-
tion by PTLC (hexanes–EtOAc = 3:1) yielded compound 32 as a yel-
5.1.18. (2S,10S,10aS)-9-Allyloxy-10-hydroxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-30-benzoyla
mino-20-triisopropylsilyloxy-30-(2-(but-3-enyloxy)phenyl)-
propanoate (30)
A solution of 23 (12 mg, 0.046 mmol, 1 equiv) in THF (1.5 mL)
was added into a stirred cold (0 °C) suspension of NaH (52.3 mg,
60%, dispersed in mineral oil, 1.3 mmol, 28 equiv) in THF (1.5 mL)
at and the resulting mixture was stirred for 10 min at 0 °C. To this
mixture was added
a
solution of 24 (22.7 mg, 0.046 mmol,
lowish oil (46 mg, 92%). ½a D23
ꢃ
ꢀ14.9 (c 1.0, CHCl3); 1H NMR
1.0 equiv) in THF (1.5 mL) and the reaction was allowed to con-
tinue for 20 min at 0 °C. Aqueous NaHCO3 (5 mL) was added and
the aqueous phase was extracted with EtOAc (10 mL ꢂ 3) and the
combined organic phase was dried and concentrated. Purification
by PTLC (hexanes–EtOAc = 3:1) yielded compound 30 as a colorless
(500 MHz) d 7.87–7.80 (m, 2H), 7.46–7.35 (m, 3H), 7.35–7.27 (m,
3H), 7.22–7.10 (m, 4H), 6.75 (d, J = 8.1 Hz, 1H), 6.62 (t, J = 9.9 Hz,
1H), 6.15–5.98 (m, 2H), 5.81 (d, J = 8.0 Hz, 1H), 5.47–5.41 (m,
1H), 5.32–5.26 (m, 2H), 5.22–5.16 (m, 1H), 5.16–5.12 (m, 1H),
4.88 (d, J = 4.2 Hz, 1H), 4.72–4.64 (m, 2H), 4.58 (m, 1H), 3.87 (d,
J = 15.0 Hz, 1H), 3.74–3.53 (m, 2H), 3.27–3.14 (m, 2H), 2.52 (m,
2H), 2.38 (m, 2H), 0.99–0.83 (m, 21H); 13C NMR (126 MHz) d
172.29, 166.26, 157.11, 137.15, 137.10, 136.82, 136.14, 134.31,
133.36, 131.57, 130.36, 128.74, 128.66, 128.60, 127.90, 127.13,
127.02, 126.96, 126.50, 126.26, 124.89, 118.95, 117.61, 116.67,
109.80, 74.79, 74.16, 69.10, 64.60, 61.52, 55.34, 53.27, 36.76,
31.69, 17.83, 17.79, 17.75, 12.46, 12.36, 0.08; HRMS (ESI) calcd
for C43H57N2O6Si m/z 725.3986 ([M+H]+), found m/z 725.3967.
oil (26 mg, 75%). ½a D23
ꢃ
ꢀ15.2 (c 1.5, CHCl3); 1H NMR (500 MHz) d
7.89–7.83 (m, 2H), 7.44–7.28 (m, 4H), 7.23–7.14 (m, 3H),
6.88–6.81 (m, 2H), 6.74 (d, J = 8.2 Hz, 1H), 6.62 (d, J = 7.7 Hz, 1H),
6.14–5.95 (m, 2H), 5.83 (dd, J = 8.5, 1.4 Hz, 1H), 5.43 (dd, J = 17.2,
1.6 Hz, 1H), 5.30–5.18 (m, 3H), 5.08 (dd, J = 13.4, 3.2 Hz, 1H), 4.97
(d, J = 1.9 Hz, 1H), 4.86 (s, 1H), 4.70–4.53 (m, 2H), 4.13–4.03 (m,
2H), 3.85 (d, J = 15.1 Hz, 1H), 3.26–3.14 (m, 2H), 2.73–2.56 (m,
2H), 2.53 (dd, J = 11.2, 6.1 Hz, 1H), 2.42 (tdd, J = 16.6, 10.2, 6.6 Hz,
2H), 2.30 (ddd, J = 12.2, 8.0, 6.0 Hz, 1H), 0.96–0.81 (m, 21H); 13C
NMR (126 MHz) d 172.47, 166.40, 157.09, 155.96, 136.13, 134.96,
134.60, 133.41, 131.50, 128.79, 128.65, 128.54, 127.59, 127.07,
126.66, 120.39, 118.92, 117.52, 117.25, 111.07, 109.81, 73.77,
73.55, 69.10, 67.47, 64.58, 61.51, 61.48, 55.37, 53.04, 33.77,
31.72, 17.83, 17.83, 17.82, 17.76, 12.31; HRMS (ESI) calcd for
5.1.21. (2S,10R,10aS)-9-Allyloxy-10-hydroxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-30-benzoyla
mino-20-triisopropylsilyloxy-30-(2-allylphenyl)-propanoate (33)
A solution of 22 (24 mg, 0.092 mmol) in THF (1.5 mL) was added
into a stirred cold (0 °C) suspension of NaH (44 mg, 60%, dispersed
in mineral oil, 1.1 mmol, 12 equiv) in THF (1.5 mL) and the result-
ing mixture was stirred for 10 min at 0 °C. To this mixture was
added a solution of 26 (43 mg, 0.093 mmol, 1.01 equiv) in THF
(1.5 mL) and the reaction was allowed to continue for 20 min at
0 °C. Aqueous NaHCO3 (5 mL) was added into the reaction mixture
and the aqueous phase was extracted with EtOAc (10 mL ꢂ 3) and
the combined organic phase was dried and concentrated. Purifica-
tion by PTLC (hexanes–EtOAc = 3:1) yielded compound 33 as a yel-
C
44H59N2O7Si m/z 755.4092 ([M+H]+), found m/z 755.4101.
5.1.19. (2S,10S,10aS)-9-Allyloxy-10-hydroxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-30-benzoyla
mino-20-triisopropylsilyloxy-30-(2-(allyloxy)phenyl)-propano
ate (31)
A solution of 23 (11.4 mg, 0.044 mmol) in THF (1.5 mL) was
added into a stirred cold (0 °C) suspension of NaH (42 mg, 60%, dis-
persed in mineral oil, 1.05 mmol, 24 equiv) in THF (1.5 mL) and the
resulting mixture was stirred for 10 min at 0 °C. To this mixture
was added a solution of 25 (22 mg, 0.046 mmol, 1.04 equiv) in
THF (1.5 mL) and the reaction was allowed to continue for
20 min at 0 °C. Aqueous NaHCO3 (5 mL) was added into the
reaction mixture and the aqueous phase was extracted with EtOAc
(10 mL ꢂ 3) and the combined organic phase was dried and con-
centrated. Purification by PTLC (hexanes–EtOAc = 3:1) yielded
lowish oil (49 mg, 73%). ½a D23
ꢃ
ꢀ8.5 (c 0.6, CHCl3); 1H NMR
(500 MHz) d 7.84–7.76 (m, 2H), 7.39–7.34 (m, 2H), 7.31–7.26 (m,
3H), 7.23–7.07 (m, 4H), 6.75 (dd, J = 10.6, 6.1 Hz, 1H), 6.64 (d,
J = 7.7 Hz, 1H), 6.14–5.99 (m, 2H), 5.79 (d, J = 8.0 Hz, 1H), 5.48–
5.41 (m, 1H), 5.37–5.28 (m, 2H), 5.19 (dd, J = 17.1, 1.6 Hz, 1H),
5.11 (dd, J = 10.1, 1.4 Hz, 1H), 4.89 (d, J = 8.5 Hz, 1H), 4.70–4.57
(m, 3H), 4.16 (s, 1H), 3.74 (d, J = 14.5 Hz, 1H), 3.70–3.54 (m, 2H),
3.29 (d, J = 14.4 Hz, 1H), 3.16 (d, J = 11.2 Hz, 1H), 2.90–2.78 (m,
1H), 2.52 (dd, J = 11.1, 5.8 Hz, 1H), 2.31 (dd, J = 16.0, 8.7 Hz, 1H),
compound 31 as a yellowish oil (29 mg, 89%). ½a D23
ꢀ14.3 (c 0.8,
ꢃ

J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
7675
2.00–1.90 (m, 1H), 1.01–0.82 (m, 21H); 13C NMR (126 MHz) d
172.07, 166.33, 137.12, 137.04, 136.93, 136.71, 134.34, 132.64,
131.45, 130.22, 128.66, 128.55, 127.99, 127.90, 127.11, 127.06,
126.97, 126.62, 126.28, 119.59, 118.61, 116.72, 109.87, 74.80,
74.12, 72.07, 69.20, 65.65, 61.13, 55.28, 53.31, 37.51, 36.84,
17.83, 17.82, 17.80, 17.79, 12.38; HRMS (ESI) calcd for
J = 15.0, 7.7 Hz, 1H), 2.37–2.19 (m, 3H), 2.14 (ddd, J = 13.4, 6.5,
4.1 Hz, 1H); 13C NMR (126 MHz)
172.05, 167.69, 166.66,
d
156.44, 156.40, 134.24, 133.17, 132.50, 131.88, 130.36, 130.19,
129.33, 128.77, 128.67, 127.37, 127.26, 127.15, 126.65, 126.55,
123.91, 120.88, 118.76, 112.45, 74.27, 71.04, 68.83, 68.52, 60.51,
57.93, 52.65, 51.23, 33.28, 32.57, 29.78; HRMS (ESI) calcd for
C
43H57N2O6Si m/z 725.3922 ([M+H]+), found m/z 725.3986.
C
40H39N2O8 m/z 675.2706 ([M+H]+), found m/z 675.2696.
5.1.22. Protected macrocyclic paclitaxel mimic 34
5.1.24. Protected macrocyclic paclitaxel mimic 35
To a stirred solution of 30 (20 mg, 0.026 mmol, 1 equiv) in
To a stirred solution of 31 (21 mg, 0.028 mmol, 1 equiv) in
CH2Cl2 (25 mL) was added
D-(ꢀ)-10-camphorsulphonic acid
CH2Cl2 (25 mL) was added
D
-(ꢀ)-10-camphorsulphonic acid
(18.5 mg, 0.079 mmol, 3 equiv) and the mixture was refluxed for
1 h. The reaction mixture was cooled to rt and Grubbs 2nd gener-
ation catalyst (2.2 mg, 10 mol %) in CH2Cl2 (5 mL) was added
slowly. The resulting solution was heated to reflux and continued
stirring for 2 h. The resulting light brown solution was washed
with 1 N aqueous NaOH (3 mL ꢂ 2), dried with anhydrous Na2SO4
and concentrated under vacuum to yield a dark brown residue. The
crude product was purified by PTLC (hexanes–EtOAc = 2:1) and the
spot with a Rf = 0.3 was collected (15.5 mg, with minor impurities)
and used in the next step without further purification.
(19.7 mg, 0.085 mmol, 3 equiv) and the mixture was stirred under
reflux for 1 h. The reaction was cooled to rt and Grubbs 2nd gener-
ation catalyst (2.4 mg, 10 mol %) in CH2Cl2 (5 mL) was added
slowly in 10 minutes. The resulting solution was heated to reflux
and continued stirring for 2 h. The resulting light brown solution
was washed with 1 N aqueous NaOH (3 mL ꢂ 2), dried with anhy-
drous Na2SO4 and concentrated under vacuum to yield a dark
brown residue. The crude product was purified by PTLC (hex-
anes–EtOAc = 2:1) and the spot with a Rf = 0.35 was collected as
the product (7 mg, with minor impurities) and used in the next
step without further purification. Also collected was the unreacted
starting material (5 mg).
The product (5 mg) after the ring-closing metathesis was dis-
solved in dry THF (1 mL) and cooled to ꢀ78 °C, followed by slow
addition of lithium bis(trimethylsilyl)amide solution in THF (1 M,
The product (7 mg) from last step was completely dried and dis-
solved in dry THF (1 mL). The resulting solution was cooled to
ꢀ78 °C, followed by slow addition of lithium bis(trimethyl-
21
l
L, 3 equiv). The resulting mixture was stirred at ꢀ78 °C for
10 min before the addition of benzoyl chloride (3.2
lL, 4 equiv)
in one portion. The reaction was allowed to continue for another
3 h at ꢀ78 °C before the addition of saturated aqueous NaHCO3
(5 mL). The resulting solution was extracted with EtOAc
(20 mL ꢂ 3), washed with brine and dried. The crude product
was purified with PTLC (5% MeOH in CH2Cl2) to yield 34 (5 mg,
silyl)amide solution in THF (1 M, 39 lL, 4 equiv). The reaction mix-
ture was stirred at ꢀ78 °C for 10 min before the addition of benzoyl
chloride (7.3 L, 8 equiv) in one portion. The reaction was allowed
l
to continue for another 3 h at ꢀ78 °C and quenched by adding sat-
urated aqueous NaHCO3 (5 mL). To the resulting mixture was
added EtOAc (20 mL). The organic solution was separated and
the aqueous was extracted with EtOAc (20 mL ꢂ 3). The combined
organic phase was washed with brine and dried. The crude product
was purified with PTLC (hexanes–EtOAc = 6:5) to yield 35 (3.4 mg,
70% from 30) as a colorless oil. ½a D23
ꢃ
ꢀ27.2 (c 0.5, CHCl3); 1H NMR
(500 MHz) d 8.23 (d, J = 7.5 Hz, 2H), 7.83 (d, J = 7.5 Hz, 2H),
7.61–7.42 (m, 6H), 7.22–7.10 (m, 3H), 7.07 (d, J = 8.5 Hz, 1H),
6.91–6.83 (m, 2H), 6.80–6.72 (m, 2H), 6.09 (d, J = 7.0 Hz, 1H),
5.92–5.78 (m, 2H), 5.57–5.45 (m, 1H), 5.35–5.25 (m, 1H), 4.91 (d,
J = 2.0 Hz, 1H), 4.42 (dd, J = 12.8, 7.9 Hz, 1H), 4.27 (dd, J = 12.7,
5.3 Hz, 1H), 3.98 (m, 1H), 3.90–3.74 (m, 3H), 3.59 (d, J = 15.2 Hz,
1H), 3.14–3.07 (m, 1H), 2.57–2.48 (m, 1H), 2.43 (m, 1H), 2.34 (m,
1H), 2.08 (m, 2H), 0.91–0.75 (m, 21H); 13C NMR (126 MHz) d
171.63, 166.70, 166.41, 155.80, 155.67, 138.04, 136.25, 134.85,
133.55, 133.13, 131.61, 130.17, 128.79, 128.58, 127.55, 127.41,
127.09, 126.95, 126.71, 124.66, 122.52, 120.71, 111.57, 73.71,
73.12, 72.04, 70.26, 68.15, 58.43, 56.04, 52.34, 51.17, 33.79,
32.83, 29.78, 17.68, 17.65, 12.27, 1.10, 1.09; HRMS (ESI) calcd for
19% for two steps from 31) as a colorless oil. ½a D23
ꢀ34.6 (c 0.3,
ꢃ
CHCl3); 1H NMR (500 MHz)
d 8.24–8.17 (m, 2H), 7.83 (d,
J = 7.3 Hz, 2H), 7.62–7.48 (m, 4H), 7.44 (t, J = 7.4 Hz, 2H), 7.24–
7.20 (m, 1H), 7.18 (d, J = 7.5 Hz, 2H), 7.13 (t, J = 7.8 Hz, 1H), 6.87
(t, J = 7.5 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 7.3 Hz, 1H),
6.73 (d, J = 8.5 Hz, 1H), 6.08 (d, J = 7.1 Hz, 1H), 5.87–5.79 (m, 2H),
5.74 (dt, J = 16.4, 3.3 Hz, 1H), 5.30 (p, J = 7.8 Hz, 1H), 4.63–4.45
(m, 4H), 4.10 (dt, J = 16.3, 2.4 Hz, 1H), 4.02–3.92 (m, 2H), 3.60 (d,
J = 15.2 Hz, 1H), 3.31 (dd, J = 9.4, 6.9 Hz, 1H), 2.69–2.55 (m, 1H),
2.16–1.94 (m, 2H), 0.87–0.76 (m, 21H); 13C NMR (126 MHz) d
171.52, 166.84, 166.64, 155.68, 155.41, 138.75, 134.62, 132.98,
131.63, 130.53, 130.04, 128.79, 128.76, 128.57, 127.70, 127.48,
127.14, 126.10, 121.01, 120.87, 120.46, 112.33, 112.03, 77.29,
73.38, 72.65, 70.94, 69.11, 65.24, 59.11, 57.14, 51.51, 51.12,
43.06, 34.30, 17.74, 17.73, 17.72, 17.70, 12.29, 0.08, 0.08, 0.07,
0.06, 0.06, 0.05; HRMS (ESI) calcd for C48H57N2O8Si m/z 817.3884
([M+H]+), found m/z 817.3815.
C
49H59N2O8Si m/z 831.4041 ([M+H]+), found m/z 831.4054.
5.1.23. Macrocyclic paclitaxel mimic 37
A solution of compound 34 (6 mg, 0.007 mmol) in THF (2 mL)
was cooled to 0 °C with an ice bath and treated with HF (0.1 mL,
70% in pyridine). The ice bath was removed and the reaction was
continued overnight. Saturated aqueous NaHCO3 (10 mL) was
added slowly and the resulting solution was extracted with EtOAc
(10 mL ꢂ 3). The EtOAc solution was washed, dried and
concentrated. The crude product was purified by PTLC (1:1
EtOAc–hexanes) to yield compound 37 (4.3 mg, 88%) as a colorless
5.1.25. Macrocyclic paclitaxel mimic 38
A solution of compound 35 (3 mg, 0.004 mmol) in THF (2 mL)
was cooled to 0 °C with an ice bath and treated with HF (0.1 mL,
70% in pyridine). The ice bath was removed and the reaction was
continued overnight. Saturated aqueous NaHCO3 (10 mL) was
added slowly and the resulting solution was extracted with EtOAc
(10 mL ꢂ 3). The EtOAc solution was washed, dried and concen-
trated. The crude product was purified by PTLC (1:1 EtOAc–hex-
anes) to yield the compound 38 (2.3 mg, 88%) as a colorless oil.
oil. ½a 2D3
ꢃ
+20.2 (c 0.5, MeOH); 1H NMR (500 MHz) d 8.27 (dd, J = 7.8,
1.8 Hz, 2H), 7.78–7.71 (m, 2H), 7.60–7.54 (m, 3H), 7.52–7.48 (m,
1H), 7.43 (t, J = 7.6 Hz, 2H), 7.31 (dd, J = 7.6, 1.4 Hz, 1H),
7.28–7.21 (m, 4H), 7.10 (t, J = 7.8 Hz, 1H), 6.94 (t, J = 7.5 Hz, 1H),
6.76 (d, J = 7.3 Hz, 1H), 6.73 (d, J = 8.3 Hz, 2H), 6.58 (d, J = 8.3 Hz,
1H), 6.26 (d, J = 6.0 Hz, 1H), 5.96 (dd, J = 8.3, 4.2 Hz, 1H), 5.81–
5.68 (m, 1H), 5.38 (dt, J = 15.6, 5.7 Hz, 1H), 5.17 (dq, J = 10.1,
5.0 Hz, 1H), 4.63 (d, J = 4.2 Hz, 1H), 4.40 (dd, J = 13.8, 5.5 Hz, 1H),
4.31 (dd, J = 13.8, 6.0 Hz, 1H), 3.82–3.70 (m, 2H), 3.66 (d,
J = 14.9 Hz, 1H), 3.49–3.38 (m, 3H), 2.89–2.76 (m, 1H), 2.44 (dt,
½
a 2D3
ꢃ
ꢀ27.5 (c 0.2, CHCl3); 1H NMR (500 MHz) d 8.35–8.28 (m,
2H), 7.79–7.73 (m, 2H), 7.57–7.38 (m, 7H), 7.25–7.22 (m, 1H),
7.11 (t, J = 7.9 Hz, 1H), 6.95 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 8.2 Hz,
1H), 6.76 (d, J = 7.3 Hz, 1H), 6.70 (t, J = 8.6 Hz, 2H), 6.14 (d,

7676
J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
J = 7.2 Hz, 1H), 6.07 (d, J = 9.1 Hz, 1H), 5.96–5.86 (m, 1H), 5.80
(d, J = 16.1 Hz, 1H), 5.36–5.26 (m, 1H), 4.62–4.45 (m, 3H), 4.33 (d,
J = 1.3 Hz, 1H), 4.16–4.06 (m, 2H), 3.96 (m, 1H), 3.62 (d,
J = 15.2 Hz, 1H), 3.24 (dd, J = 9.2, 6.5 Hz, 1H), 2.71–2.63 (m, 1H),
2.27 (dt, J = 14.3, 8.1 Hz, 1H), 2.22–2.17 (m, 1H); 13C NMR
(126 MHz) d 173.03, 166.96, 166.45, 155.70, 155.33, 134.55,
132.86, 131.66, 130.65, 130.24, 129.16, 128.65, 128.48, 128.21,
127.95, 127.35, 127.24, 127.19, 126.42, 121.28, 121.14, 120.30,
112.91, 112.20, 74.00, 72.25, 70.46, 68.30, 66.14, 59.10, 56.62,
+42.5 (c 0.2, CHCl3); 1H NMR (500 MHz) d 8.19 (d, J = 8.0 Hz, 2H),
7.75 (d, J = 14.8 Hz, 1H), 7.63 (dt, J = 9.0, 4.5 Hz, 3H), 7.52 (t,
J = 7.7 Hz, 2H), 7.49–7.42 (m, 1H), 7.37 (t, J = 7.7 Hz, 2H),
7.13–7.07 (m, 1H), 6.89 (s, 1H), 6.65 (s, 1H), 6.48–6.39 (m, 2H),
6.19 (d, J = 4.5 Hz, 1H), 5.90–5.76 (m, 1H), 5.59–5.50 (m, 1H),
5.39 (d, J = 9.2 Hz, 1H), 4.86 (s, 1H), 4.39 (dd, J = 14.3, 7.3 Hz, 1H),
4.08 (d, J = 6.7 Hz, 1H), 4.01 (d, J = 14.2 Hz, 1H), 3.89 (s, 1H),
3.72–3.44 (m, 3H), 3.21 (d, J = 14.8 Hz, 2H), 2.99 (s, 1H), 2.51 (s,
2H); HRMS (ESI) calcd for C39H37N2O7 m/z 645.2595 ([M+H]+),
found m/z 645.2570.
51.20, 48.91, 34.17; HRMS (ESI) calcd for
C39H37N2O8 m/z
661.2550 ([M+H]+), found m/z 661.2485.
5.1.28. (2S,10R,10aS)-9-Allyloxy-10-benzoyloxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-30-benzoyla
mino-20-triisopropylsilyloxy-30-(2-allylphenyl)-propanoate (42)
A solution of 33 (8 mg, 0.011 mmol) in dry THF (2 mL) was
cooled to ꢀ78 °C. To this solution was added slowly lithium bis(tri-
5.1.26. Protected macrocyclic paclitaxel mimic 36
To a stirred solution of 32 (25 mg, 0.034 mmol, 1 equiv) in
CH2Cl2 (25 mL) was added
D
-(ꢀ)-10-camphorsulphonic acid
(24 mg, 0.103 mmol, 3 equiv) and the mixture was stirred under
reflux for 1 h. The reaction was cooled to rt and Grubbs 2nd gener-
ation catalyst (1.5 mg, 5 mol %) in CH2Cl2 (5 mL) was added slowly
in 10 minutes. The resulting solution was heated to reflux and con-
tinued overnight. The resulting light brown solution was washed
with 1 N aqueous NaOH (3 mL ꢂ 2), dried with anhydrous Na2SO4
and concentrated under vacuum to yield a dark brown residue. The
crude product was purified by PTLC (hexanes–EtOAc = 3:1) and the
spot with a Rf = 0.3 was collected as the product (3.3 mg, with min-
or impurities) and used in the next step without further purifica-
tion. Also collected was the unreacted starting material (10 mg).
The product (3.3 mg) from last step was completely dried and
dissolved in dry THF (1 mL). The resulting solution was cooled to
ꢀ78 °C, followed by slow addition of lithium bis(trimethyl-
methylsilyl)amide solution in THF (1 M, 44 lL, 4 equiv). The reac-
tion mixture was stirred at ꢀ78 °C for 10 min before the addition
of benzoyl chloride (11 L, 8 equiv) in one portion. The reaction
l
was allowed to continue for another 3 h at ꢀ78 °C and quenched
by adding saturated aqueous NaHCO3 (5 mL). To the resulting mix-
ture was added EtOAc (20 mL). The organic solution was separated
and the aqueous was extracted with EtOAc (20 mL ꢂ 3). The com-
bined organic phase was washed with brine and dried. The crude
product was purified with PTLC (hexanes–EtOAc = 4:1) to yield
42 (5.5 mg, 60%) as a colorless oil. ½a D23
ꢃ
ꢀ20.9 (c 0.6, CHCl3); 1H
NMR (500 MHz) d 7.99 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 7.9 Hz, 2H),
7.51 (t, J = 7.3 Hz, 1H), 7.38 (dd, J = 10.2, 4.9 Hz, 3H), 7.33 (t,
J = 7.3 Hz, 1H), 7.28–7.17 (m, 8H), 7.16–7.09 (m, 1H), 6.72 (d,
J = 8.2 Hz, 1H), 6.63 (d, J = 7.6 Hz, 1H), 6.45 (d, J = 8.2 Hz, 1H),
6.08 (ddt, J = 16.7, 10.2, 6.3 Hz, 1H), 5.79 (d, J = 8.0 Hz, 1H), 5.66
(ddt, J = 16.5, 10.7, 5.5 Hz, 1H), 5.27–5.14 (m, 3H), 5.07 (d,
J = 17.3 Hz, 1H), 4.96 (d, J = 10.5 Hz, 1H), 4.67 (s, 1H), 4.36 (ddd,
J = 60.8, 12.4, 5.4 Hz, 2H), 3.77–3.55 (m, 3H), 3.34 (d, J = 14.3 Hz,
1H), 3.14 (d, J = 11.3 Hz, 1H), 2.73–2.61 (m, 1H), 2.58–2.40 (m,
2H), 1.03–0.77 (m, 21H); 13C NMR (126 MHz) d 172.28, 166.37,
165.98, 157.69, 138.03, 137.13, 136.66, 134.29, 132.82, 132.67,
131.42, 130.87, 130.33, 130.16, 129.84, 128.74, 128.52, 128.43,
128.20, 127.91, 127.05, 126.96, 126.26, 122.76, 118.84, 117.61,
116.84, 110.04, 75.01, 73.73, 71.57, 69.12, 65.21, 60.98, 60.49,
54.94, 53.38, 36.91, 36.82, 21.13, 17.83, 17.80, 14.28, 12.40; HRMS
(ESI) calcd for C43H57N2O6Si m/z 829.4248 ([M+H]+), found m/z
829.4201.
silyl)amide solution in THF (1 M, 19
ture was stirred at ꢀ78 °C for 10 min before the addition of benzoyl
chloride (4.4 L, 8 equiv) in one portion. The reaction was allowed
lL, 4 equiv). The reaction mix-
l
to continue for another 3 h at ꢀ78 °C and quenched by adding sat-
urated aqueous NaHCO3 (5 mL). To the resulting mixture was
added EtOAc (20 mL). The organic solution was separated and
the aqueous was extracted with EtOAc (20 mL ꢂ 3). The combined
organic phase was washed with brine and dried. The crude product
was purified with PTLC (hexanes–EtOAc = 1:1) to yield 36 (1.5 mg,
9% for two steps from 32) as a colorless oil. ½a D23
ꢃ
+11.0 (c 0.3,
CHCl3); 1H NMR (500 MHz) d 8.30–8.26 (m, 2H), 7.83–7.79 (m,
2H), 7.59–7.46 (m, 4H), 7.46–7.40 (m, 2H), 7.39–7.35 (m, 1H),
7.19–7.07 (m, 4H), 7.01 (dd, J = 7.2, 1.7 Hz, 1H), 6.78–6.74 (m,
2H), 6.05 (d, J = 7.4 Hz, 1H), 5.95–5.88 (m, 1H), 5.83–5.75 (m,
1H), 5.65 (dd, J = 8.1, 1.7 Hz, 1H), 5.30–5.21 (m, 1H), 4.52 (dd,
J = 13.7, 6.6 Hz, 1H), 4.33 (d, J = 1.9 Hz, 1H), 4.19 (dd, J = 14.3,
4.1 Hz, 1H), 4.05–3.94 (m, 2H), 3.61 (d, J = 15.4 Hz, 1H), 3.46 (d,
J = 7.0 Hz, 2H), 3.20 (dd, J = 10.1, 5.8 Hz, 1H), 2.62–2.53 (m, 1H),
2.28 (dd, J = 9.8, 7.1 Hz, 1H), 2.10 (m, 1H), 1.08–0.79 (m, 21H);
13C NMR (126 MHz) d 171.30, 166.87, 166.51, 155.35, 137.58,
137.23, 135.02, 134.67, 133.10, 132.93, 131.58, 130.45, 130.25,
129.95, 129.87, 128.73, 128.61, 128.15, 127.64, 127.26, 127.04,
126.96, 126.49, 126.43, 123.37, 120.63, 117.31, 74.61, 73.39,
70.23, 68.48, 59.30, 57.28, 52.37, 51.53, 35.72, 31.68, 17.86,
17.82, 12.40; HRMS (ESI) calcd for C48H57N2O7Si m/z 801.3935
([M+H]+), found m/z 801.3917.
5.1.29. (2S,10R,10aS)-9-Allyloxy-10-benzoyloxy-1,2,3,5,10,10a-
hexahydropyrrolo[1,2-b]isoquinolin-2-yl (20S,30R)-30-benzoyla
mino-20-hydroxy-30-(2-allylphenyl)-propanoate (43)
A solution of compound 42 (3.3 mg, 0.004 mmol) in THF (3 mL)
was cooled to 0 °C with an ice bath and treated with HF (0.1 mL,
70% in pyridine). The ice bath was removed and the reaction was
continued overnight. Saturated aqueous NaHCO3 (5 mL) was added
slowly and the resulting solution was extracted with EtOAc
(10 mL ꢂ 3). The EtOAc solution was washed, dried and concen-
trated. The crude product was purified by PTLC (50% EtOAc in hex-
anes) to yield compound 43 (2.4 mg, 89%) as a colorless oil. ½a D23
ꢃ
+2.0 (c 0.3, CHCl3); 1H NMR (500 MHz) d 8.00 (d, J = 8.0 Hz, 2H),
7.75 (d, J = 8.0 Hz, 2H), 7.52 (dd, J = 11.5, 5.0 Hz, 2H), 7.39 (tt,
J = 26.5, 7.8 Hz, 6H), 7.25–7.19 (m, 3H), 6.89 (d, J = 8.6 Hz, 1H),
6.72 (d, J = 8.0 Hz, 2H), 6.52 (d, J = 8.2 Hz, 1H), 6.06 (tt, J = 16.5,
6.2 Hz, 1H), 5.94 (dd, J = 8.6, 2.2 Hz, 1H), 5.64 (qd, J = 10.7, 5.5 Hz,
1H), 5.27 (t, J = 8.5 Hz, 1H), 5.17–5.01 (m, 3H), 4.95 (d,
J = 10.5 Hz, 1H), 4.56 (d, J = 2.2 Hz, 1H), 4.34 (ddd, J = 17.9, 12.3,
5.1 Hz, 2H), 3.92 (d, J = 14.4 Hz, 1H), 3.69–3.53 (m, 2H), 3.46 (d,
J = 14.3 Hz, 1H), 3.29 (d, J = 11.3 Hz, 1H), 2.80–2.67 (m, 1H), 2.62–
2.50 (m, 2H), 2.33 (t, J = 7.5 Hz, 1H), 2.30–2.22 (m, 1H); 13C NMR
5.1.27. Macrocyclic paclitaxel mimic 39
A solution of compound 36 (1.5 mg, 0.002 mmol) in THF (2 mL)
was cooled to 0 °C with an ice bath and treated with HF (0.1 mL,
70% in pyridine). The ice bath was removed and the reaction was
continued overnight. Saturated aqueous NaHCO3 (10 mL) was
added slowly and the resulting solution was extracted with EtOAc
(10 mL ꢂ 3). The EtOAc solution was washed, dried and concen-
trated. The crude product was purified by PTLC (75% EtOAc in hex-
anes) to yield compound 39 (1 mg, 83%) as a colorless oil. ½a D23
ꢃ

J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
7677
(126 MHz) d 172.94, 166.88, 165.99, 157.75, 138.21, 137.90,
137.08, 136.85, 134.15, 132.75, 132.72, 131.66, 130.81, 130.56,
129.85, 128.89, 128.61, 128.34, 128.23, 127.19, 126.95, 122.76,
118.80, 117.64, 116.71, 110.20, 74.90, 73.14, 71.65, 69.12, 65.39,
60.75, 55.23, 51.56, 37.25, 33.55, 32.01, 24.82, 22.78, 14.21, 0.08;
HRMS (ESI) calcd for C43H57N2O6Si m/z 673.2914 ([M+H]+), found
m/z 673.2876.
5.7 Hz, 2H), 5.36 (s, 1H), 4.55 (d, J = 5.3 Hz, 2H), 4.37 (d,
J = 16.0 Hz, 1H), 4.28 (s, 1H), 3.77 (d, J = 15.8 Hz, 1H), 3.62 (dd,
J = 14.6, 6.1 Hz, 2H), 3.49–3.31 (m, 2H), 2.86 (d, J = 11.6 Hz, 1H),
2.46 (dt, J = 14.8, 7.5 Hz, 1H), 2.15 (d, J = 14.2 Hz, 1H); 13C NMR
(126 MHz) d 172.46, 168.48, 166.92, 157.58, 137.43, 137.34,
134.03, 133.71, 133.12, 131.81, 130.61, 130.36, 129.88, 129.45,
128.60, 128.59, 128.40, 128.37, 127.39, 127.21, 126.36, 120.01,
114.46, 76.20, 72.89, 69.94, 68.15, 62.50, 58.17, 54.20, 51.21,
37.95, 36.09; HRMS (ESI) calcd for C39H37N2O7 m/z 645.2601
([M+H]+), found m/z 645.2573.
5.1.30. Protected macrocyclic paclitaxel mimic 40
To a solution of 33 (48 mg, 0.066 mmol) in CH2Cl2 (50 mL) was
added
D
-(ꢀ)-10-camphorsulfonic acid (76.6 mg, 0.33 mmol,
5 equiv) and the mixture was stirred under reflux for 1 h. The reac-
tion was cooled to rt and Grubbs 2nd generation catalyst (5.6 mg,
10 mol %) in CH2Cl2 (5 mL) was added slowly in 10 min. The result-
ing solution was heated to reflux and continued stirring overnight.
The resulting light brown solution was washed with 1 N aqueous
NaOH (3 mL ꢂ 2), dried with anhydrous Na2SO4 and concentrated
under vacuum to yield a dark brown residue. The crude product
was purified by PTLC (hexanes–EtOAc = 1:2) to yield the bridged
compound with a Rf = 0.25 (16 mg) together with unreacted start-
ing material (12 mg).
5.2. X-ray methods
5.2.1. X-ray of compound 22
A colorless prism (0.07 ꢂ 0.07 ꢂ 0.08 mm3) was centered on the
goniometer of an Oxford Diffraction Gemini A Ultra diffractometer
operating with CuK
a radiation. The data collection routine, unit
cell refinement, and data processing were carried out with the pro-
gram CrysAlisPro.36 The Laue symmetry and systematic absences
were consistent with the monoclinic space groups P21 and P21/m.
As the substance was enantiomerically pure, the acentric space
group P21 was chosen. The structure was solved by direct methods
and refined using SHELXTL NT.37 The asymmetric unit of the struc-
ture comprises two crystallographically independent molecules.
The final refinement model involved anisotropic displacement
parameters for non-hydrogen atoms. A riding model was used for
the aromatic and alkyl hydrogens. The hydrogen atom positions
of the hydroxyl groups were located and refined independently.
The absolute configuration was established from anomalous dis-
persion effects (Flack x = 0.0(2)). [Flack x = XXXX;38 Hooft
P2(true) = 1.000, P3(true) = 1.000, P3(rac-twin) = 0.000; P3(false) =
0.000, y = ꢀ0.08(11)].24,39
The product (8 mg, 0.012 mmol) from the previous step was
completely dried and dissolved in dry THF (2 mL). The resulting
solution was cooled to ꢀ78 °C, followed by slow addition of lithium
bis(trimethylsilyl)amide solution in THF (1 M, 46 lL, 4 equiv). The
reaction mixture was stirred at ꢀ78 °C for 10 min before the addi-
tion of benzoyl chloride (10.7 L, 8 equiv) in one portion. The
l
reaction was allowed to continue for another 3 h at ꢀ78 °C and
quenched by adding saturated aqueous NaHCO3 (5 mL). To the
resulting mixture was added EtOAc (20 mL). The organic solution
was separated and the aqueous was extracted with EtOAc
(20 mL ꢂ 3). The combined organic phase was washed with brine
and dried. The crude product was purified with PTLC (hexanes–
EtOAc = 1:2) to yield 40 (7.6 mg, 39% for two steps from 33) as a
5.2.2. X-ray of compound 23
colorless oil. ½a D23
ꢃ
ꢀ11.3 (c 0.6, CHCl3); 1H NMR (500 MHz) d 8.00
A colorless rod (0.05 ꢂ 0.13 ꢂ 0.29 mm3) was centered on the
(d, J = 7.9 Hz, 2H), 7.77 (d, J = 7.6 Hz, 2H), 7.55–7.46 (m, 2H), 7.41
(dt, J = 15.7, 7.7 Hz, 4H), 7.30 (dt, J = 16.7, 8.3 Hz, 2H), 7.23 (t,
J = 7.9 Hz, 1H), 7.21–7.09 (m, 3H), 6.80 (t, J = 8.7 Hz, 2H), 6.62 (t,
J = 11.8 Hz, 2H), 5.66 (d, J = 7.9 Hz, 1H), 5.37 (d, J = 15.9 Hz, 1H),
5.27 (t, J = 5.3 Hz, 1H), 4.74 (d, J = 14.3 Hz, 1H), 4.41–4.20 (m,
3H), 4.02 (dd, J = 16.5, 7.1 Hz, 1H), 3.75 (d, J = 15.8 Hz, 1H), 3.65–
3.45 (m, 2H), 3.41 (dd, J = 11.5, 6.0 Hz, 1H), 2.73 (t, J = 15.8 Hz,
1H), 2.54 (dt, J = 14.5, 7.2 Hz, 1H), 2.44–2.32 (m, 1H), 0.89–0.61
(m, 21H); 13C NMR (126 MHz) d 171.75, 166.32, 166.28, 158.91,
138.05, 137.84, 137.19, 134.51, 132.78, 131.58, 131.32, 130.89,
130.69, 129.90, 129.58, 128.71, 128.30, 128.11, 127.78, 127.72,
127.08, 126.32, 123.60, 120.84, 114.37, 76.90, 74.50, 70.60, 67.83,
65.24, 57.75, 52.79, 50.23, 38.57, 34.81, 17.72, 17.69, 12.33; HRMS
(ESI) calcd for C48H57N2O7Si m/z 801.3935 ([M+H]+), found m/z
801.3883.
goniometer of an Oxford Diffraction Gemini A Ultra diffractometer
operating with CuK
a radiation. The data collection routine, unit
cell refinement, and data processing were carried out with the pro-
gram CrysAlisPro.36 The Laue symmetry was consistent with the
triclinic space groups P1 and Pꢀ1. As the substance was enantio-
merically pure, the acentric space group P1 was chosen. The struc-
ture was solved by direct methods and refined using SHELXTL
NT.37 The asymmetric unit of the structure comprises two crystal-
lographically independent molecules. The final refinement model
involved anisotropic displacement parameters for non-hydrogen
atoms. A riding model was used for the alkyl hydrogens. The
hydrogen atom positions of the hydroxyl groups were located
and refined independently. The absolute configuration was estab-
lished from anomalous dispersion effects [Flack x = 0.02(14)38
;
Hooft P2(true) = 1.000, P3(true) = 1.000, P3(rac-twin) = 0.000;
P3(false) = 0.000, y = ꢀ0.05(5)].24,39
5.1.31. Macrocyclic paclitaxel mimic 41
A solution of compound 40 (5.5 mg, 0.007 mmol) in THF (3 mL)
was cooled to 0 °C with an ice bath and treated with HF (0.1 mL,
70% in pyridine). The ice bath was removed and the reaction was
continued overnight. Saturated aqueous NaHCO3 (10 mL) was
added slowly and the resulting solution was extracted with EtOAc
(10 mL ꢂ 3). The EtOAc solution was washed, dried and concen-
trated. The crude product was purified by PTLC (3:1 EtOAc–
hexanes) to yield compound 41 (3.5 mg, 85%) as a colorless oil.
5.3. Biological experiments
5.3.1. Antiproliferative activities
Antiproliferative activity against the A2780 cell line was deter-
mined as described previously.40
5.3.2. Tubulin polymerization assays
½
a 2D3 +2.6 (c 0.3, CHCl3); 1H NMR (500 MHz) d 8.03–7.96 (m, 2H),
ꢃ
5.3.2.1. Absorption.
Tubulin assembly was monitored by
measuring optical density at 350 nm using a Hewlett–Packard
8453 absorption spectrophotometer. Bovine brain tubulin (final
concentration 15 lM) in PMEG buffer (100 mM Pipes, 1 mM
MgSO4, 2 mM EGTA, 1 mM GTP, pH 6.9) was equilibrated to 37 °C
in the sample cell and a baseline was recorded. Taxol (final concen-
7.83 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 7.7 Hz, 1H), 7.66 (d, J = 7.5 Hz,
1H), 7.57–7.50 (m, 1H), 7.45 (dd, J = 11.1, 3.8 Hz, 1H), 7.40 (t,
J = 7.3 Hz, 2H), 7.34 (t, J = 7.2 Hz, 2H), 7.24 (d, J = 4.0 Hz, 2H), 7.19
(t, J = 7.9 Hz, 1H), 7.12 (d, J = 6.0 Hz, 1H), 6.78 (d, J = 7.9 Hz, 2H),
6.59 (d, J = 5.4 Hz, 1H), 6.07–5.97 (m, 1H), 5.55 (dd, J = 15.6,

7678
J. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 7664–7678
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DMSO was added and optical density was monitored until a steady
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sembly. The control contained tubulin and DMSO but no ligand.
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Tubulin assembly was monitored by
observing the increase in DAPI fluorescence as a function of time
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excess GDP (final concentration 5 mM). Excess GDP was removed
by rapid gel filtration into PMED buffer (PME buffer with 0.1 mM
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GDP). Samples containing tubulin (final concentration 8
l
M) and
40,6-diamidino-2-phenylindole (DAPI) (final concentration 10
l
M)
in PMED buffer was equilibrated to 37 °C in the 96-well black plate
(Nunc). The excitation and emission wavelengths were set at 360
and 450 nm, respectively, and the fluorescence was recorded to ob-
tain a baseline. Taxol (final concentration 8
lM) or test compound
(final concentration 60 M) in DMSO was added to initiate poly-
l
merization. Fluorescence was monitored until a steady state was
reached. The control contained tubulin, DAPI, and DMSO but no li-
gand. The final concentration of DMSO was 10% (v/v) in all
samples.
Acknowledgments
This work was supported by NIH grant CA-69571; we are grate-
ful for this support. We thank William Bebout (Virginia Tech) for
mass spectroscopic determinations, and Peggy Brodie (Virginia
Tech) for A2780 cytotoxicity assays.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.10.010. These data
include MOL files and InChiKeys of the most important compounds
described in this article. CCDC 850019 and 850020 contain the sup-
plementary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via http://www.ccdc.cam.ac.uk/data_request/cif.
24. Spek, A. L. J. Appl. Cryst. 2003, 36, 7.
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