Spirobicyclo[2.2.2]octane derivatives: Mimetics of baccatin III and paclitaxel (Taxol)

Article abstract of DOI:10.1039/b409678a

The formylated spirobyclic alcohol 8a was computer modeled to be a mimetic of paclitaxel. In this model, the formyl group was used as a truncated paclitaxel side chain in order to reduce the computational work. Compound 8c, carrying the paclitaxel side chain, was synthesized in six steps from optically active 1,3-diketone 12. Microtubule stabilization was not observed for 8c, indicating that the model needs to be adjusted.

Full text of DOI:10.1039/b409678a

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A R T I C L E
Spirobicyclo[2.2.2]octane derivatives: mimetics of baccatin III and
paclitaxel (Taxol)†‡
Fredrik Almqvist,a Sophie Manner,b Viveca Thornqvist,b Ulf Berg,b Margareta Wallinc and
Torbjörn Frejd*b
a
Department of Organic Chemistry, Umeå University, SE 901 87 Umeå, Sweden
The Laboratory of Organic and Bioorganic Chemistry, Department of Chemistry,
b
Lund University, P. O. Box 124, SE 221 00 Lund, Sweden. E-mail: Torbjorn.Frejd@orgk1.lu.se
Department of Zoology, Göteborg University, P.O. Box 463, SE 405 30 Göteborg, Sweden
c
Received 25th June 2004, Accepted 3rd September 2004
First published as an Advance Article on the web 6th October 2004
The formylated spirobyclic alcohol 8a was computer modeled to be a mimetic of paclitaxel. In this model, the
formyl group was used as a truncated paclitaxel side chain in order to reduce the computational work. Compound
8c, carrying the paclitaxel side chain, was synthesized in six steps from optically active 1,3-diketone 12. Microtubule
stabilization was not observed for 8c, indicating that the model needs to be adjusted.
Structure activity data on paclitaxel analogs indicate that
Introduction
several functional group variations are allowed at the northern
half without seriously affecting the microtubule stabilization.
Thus, one would expect that a “Taxol mimetic” would not
strictly have to mimic these parts of the molecule. It seems
more critical, however, that the side-chain and in particular the
2-hydroxyl is properly positioned. Also the 2-benzoyloxy, the
4-acetoxy and the oxetane moieties seem to be necessary for
the activity of Taxol and related compounds. This aspect has
recently been discussed.19,20
Paclitaxel (Taxol) 1a and docetaxel (Taxotere) 1b (Fig. 1), are
well known microtubule stabilizing anticancer agents1,2 and
paclitaxel has been prepared by several independent total
synthetic routes.3–10 However, ingenious as they may be, these
syntheses require many and complex synthetic steps. Indeed, the
diterpenoid core of these bio-active molecules, i.e. baccatin III
and its derivatives, is still a major synthetic challenge.
Several compounds of widely varying structures show micro-
tubule activity and there seems to be considerable opportunity
to develop alternative structures having taxane-like properties.
Since the taxane diterpenoid skeleton (baccatin III) is quite rigid
and rather difficult to synthesize, we looked for simple rigid
replacements of this structure that might be easier to synthe-
size and, decorated with the necessary pharmacophores, would
possibly have the same or similar biological activity as paclitaxel
itself. Moreover, in the long run it is necessary to develop new
alternatives to taxoid anticancer drugs due to the occurrence of
multidrug resistance.21
Results and discussion
We first made a computer investigation to establish which
conformation of paclitaxel/docetaxel would be most suitable
as a template for comparison of new structures. Preferably, this
would be the bioactive conformation. Nogales and co-workers
have determined the structure of the a,b-tubulin heterodimer
(the basic structural unit of microtubule) bound to paclitaxel.
A resolution of 3.7 Å was obtained by electron crystallography
of zinc-induced tubuline sheets.22,23 This apparently shows the
location of the binding site of paclitaxel. However, the struc-
ture lacks the degree of resolution to allow the determination
of a more detailed bioactive conformation. Therefore, we
decided to use crystal structures of paclitaxel and docetaxel,
since these must be low energy conformations. A crystal struc-
ture of docetaxel was reported by Gueritte in 199024 and later
Mastropaolo published the crystal structures of two different
conformers of paclitaxel, A and B.25 In order to determine
which one to apply as our template we made an overlay of these
structures in which we also included our own energy minimised
version of paclitaxel (Fig. 3). The Macromodel computer
program was used for this purpose.26
Fig. 1
In addition to paclitaxel, docetaxel and modified taxanes,
e.g. 2,11 3,12 413 and 5,14 a number of non-taxoids have been
shown to be microtubule stabilizers (Fig. 2). Thus, the bicyclic
epoxide 6 was reported to be a microtubule stabilizer.15
Howarth et al. reported microtubule stabilization of guanosine
derivative 7 carrying the paclitaxel side chain (R in formula 1a).16
Very recently, Snyder et al. showed that a taxane derivative 5,
lacking the oxetane ring, was potent as a microtubule stabilizer
in vitro, but had a much lower cytotoxicity than paclitaxel.14
It should also be noted that a number of other non-taxanes
have been found to be microtubule stabilizers (epothilones,
sarcodictyin, synstab A, jatrophanes and others).17,18
† Electronic supplementary information (ESI) available: Table of
1
results of the alkylation of compound 12, copies of the H NMR and
13C NMR spectra for all compounds, NOESY spectra for compounds
13a, 13b and the O-alkylated major products, the overlay between 8b
and paclitaxel A and the overlay of the different conformers of 8a/8b.
See http://www.rsc.org/suppdata/ob/b4/b409678a/
As can be seen, there are no major differences between the
rigid cores and it is almost exclusively the side chain that differs
in its orientations. Snyder et al. have addressed this problem
and suggested, by using both theoretical and experimental
‡ Taken in part from the PhD thesis of Fredrik Almqvist, Lund
University, 1996.
T h i s j o u r n a l i s
©
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 8 5 – 3 0 9 0
3 0 8 5

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Fig. 3 Stereoview of overlay containing crystal structure of docetaxel,
crystal structures of two different conformers of paclitaxel, A and B,
and the calculated minimum energy conformation of paclitaxel.
Color code: green = docetaxel; blue = paclitaxel A, red = paclitaxel B;
magenta = energy minimized paclitaxel.
would be interesting candidates and the best fit to the taxane
diterpenoid core was found for 8 (Fig. 4).32 In fact, Kuwajima
et al. indicated that spirobicyclics would constitute a new lead
to taxane-like biologically active compounds and also that
one of their compounds obtained by an unexpected Cope-
rearrangement of a tricyclic framework, 9, exhibited the same
potency as verapamil as a MDR (multi drug resistant) reversing
agent.33
Fig. 4
Further computational work was made by use of the
MacroModelcomputerprogram.26 Thus,theindividualcandidate
structures were energy minimised (for details see Experimental).
In order to simplify the calculations the paclitaxel side chain was
replacedbyaformicester.Comparisonof paclitaxelAandseveral
spirobicyclic structures (overlay) resulted in the identification of
8a as a fair mimetic of the southern part of paclitaxel A. In these
overlays the highlighted six atoms shown in Fig. 5 were used as
contact points to comply with the pharmacophore model (13-O,
2-O, 4-O, and the oxetane ring, as mentioned).
Fig. 2 Examples of structures showing biological activity comparable
to paclitaxel.
evidence that it is the T-paclitaxel conformation that fits into
the b-tubulin binding site.27–29 It is of particular interest that it
is not the close contact between the phenyl groups emanating
from the 3-position and the 2-O-benzoate (hydrophobically
collapsed conformer) that is important but the rather close
contact between the former and the 4-OAc methyl group.29
However, along with the suggestion of Swindell et al.30 we
reasoned that the side chain should have the same or similar
degrees of freedom to orient itself in all of these and also in new
structures. Thus, we concluded that any of the above structures
were useful as our template for the taxane core and we decided
to use paclitaxel A.
Fig. 5 Overlay between paclitaxel A and 8a, rms: 0.415.
When performing the energy minimisations of the mimetics
another conformer of 8a was found, namely 8b, which had
approximately the same steric energy as 8a. However, a much
worse overlay rms value was obtained for 8b/paclitaxel
A (rms 0.729) than for 8a/paclitaxel A (rms 0.415). The difference
in geometry of 8a and 8b is a flip of the six-membered spiro ring
A number of MM3-energy minimised rigid structural motifs
were then checked for structural similarity with paclitaxel
A by using the MacMimic computer program.31 Based on
this preliminary test it appeared that spirobicyclo frameworks
3 0 8 6
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 8 5 – 3 0 9 0

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(Fig. 6). With a calculated energy difference of 0.01 kcal mol−1
between 8a and 8b, they would approximately equally populate
the equilibrium conformations. We assume that the ring flip
should have a low energy barrier, thus, allowing the best fitting
structure, 8a, to be attached to the microtubule binding-site.
Fig. 6 Stereoview of the overlays of 8a and 8b. 8a: spiro ring in back
position (blue), 8b: spiro ring in front position (magenta).
In the synthetic planning of 8c (carrying the paclitaxel side
chain) we imagined that bicyclo[2.2.2]octane-2,6-dione could
be used as a starting material, since it was available to us in
substantial amounts for other projects.34–36 Thus, the class
of spirobicyclo derivatives such as 8 appeared to be easy to
synthesize and even transformable into 10 (Scheme 1) or closely
related compounds. A few other spirobicyclic derivatives of
similar structures have been synthesized earlier,33,37–40 but these
compounds can not easily be used for our purpose.
We here report a six step synthesis of optically active 8c,
starting from 12, which was prepared from easily available
optically active (>96% ee) keto-alcohol 1134 (Scheme 1).
Alkylation of 12 with 2-(2-iodo)-1,3-dioxolane using Cs2CO3
as a base provided 13a, which carried the necessary function-
alities and number of carbon atoms for the subsequent aldol
cyclization. Preference for alkylation of the exo-face was
achieved due to the steric influence of the endo-acetoxy group,
although to a lesser extent than we hoped. The exo:endo ratio
in the best case was 80:20, which was not improved by the use
of the corresponding TBDMS-protected derivative.41 In addi-
tion to the C-alkylation products substantial amounts of two
O-alkylation products were formed in all cases, despite the use
of several methods that were recommended to avoid reactions
at oxygen.42–46 Hydrolysis of the two O-alkylation products
regenerated 12 but only product 13a was used for further
synthesis. Fortunately, column chromatography allowed purifi-
cation of 13a, which was isolated in 45% yield. A NOESY cross
peak due to the proximity of the methyl group of the acetyl
group and the endo-5H (indicated in Scheme 1 with an arrow)
clearly supported the structure of 13a. Several other combina-
tions of bases and solvents were used in order to improve the
selectivity without success. The C/O alkylation ratios varied
between 44:56 and 67:33 and the exo:endo ratios between
64:36 and 80:20 (see Electronic Supplementary Informa-
tion†). To avoid the O-alkylation problem we also applied a
Mukaiyama-type reaction47,48 between the TMS-enol ethers of
12 (mixture of isomers)49 and the dimethyl acetal of acrolein,
but without success. It only resulted in the recovery of 12.
Aqueous acid treatment of 13a could conceivably lead to
hydrolysis of the two protective groups (the acetal and the
acetate) followed directly by an acid catalyzed aldol cyclization
to give the spiro-anellated cyclohexenone ring of 15. However,
neither the reaction conditions (1 M HCl, THF), that we had
previously used for a similar purpose,50 nor several other acidic
reagents (PPTS in acetone water, wet silica, oxalic acid in
dichloromethane and PdCl2(CH3CN)2 (cat.) in wet acetone51)
were effective in hydrolyzing the acetal unit. Curiously,
treatment of 13a with 1 M HCl in acetone did result in a slow
conversion into the corresponding aldehyde (60%) but the
desired cyclization was not observed. Stronger acid and higher
temperature destroyed the material. An efficient method for
acetal hydrolysis was finally found to be treatment of 13a with
80% acetic acid at 65 °C. Even though these conditions did not
lead to a combined aldol cyclization and acetate hydrolysis, an
almost quantitative yield of the corresponding aldehyde was
obtained. The aldol cyclization to give 14 was then performed
simply by changing the medium to toluene–TsOH (cat.)
Scheme 1 (a) Cs2CO3, 2-(2-iodoethyl)-1,3-dioxolane, toluene, 100 °C,
3 h, 13b (9%):13a (45%) 20:80 and two O-alkylation products; (b) acetic
acid (80%), 65 °C, 3 h; (c) TsOH, toluene, Soxhlet apparatus (4 Å
molecular sieves), reflux, 3 h, 85% from 13a; (d) K2CO3, MeOH:H2O
9:1, room temperature, 2 h, 86%; (e) DMAP, DCC, 17, CH2Cl2, 0 °C
to room temperature, 3 h, 55%; (f) Me2BBr, CH2Cl2, −70 °C to −50 °C,
2 h, 70%.
followed by azeotropic removal of water. Subsequent basic ester
hydrolysis of 14 gave 15 in 73% yield over three steps.
Next, the side chain of paclitaxel, in the form of (2R,3S)-
N-benzoyl-2-O-(methoxymethyl)-phenylisoserine (17), avail-
able in our laboratory from earlier work in the taxane field,52
was attached to 15 by DCC/DMAP treatment according to
an earlier report by Greene et al. for similar cases.53 Finally, 8c
was obtained after removal of the MOM protecting group by
Me2BBr54 in 39% yield over two steps. Some of the 2-epimer
(ca. 10% of the yield) was also formed, but was removed by
chromatographic separation (HPLC).
Compound 8c dissolved in DMSO:water 25:75 was
submitted to a microtubule polymerization assay but did not
show any microtubule stabilization. Microtubule proteins
(3 mg ml−1) were assembled at 30 °C in the presence or absence
of 0.305 or 0.61 mM compound 8c dissolved in DMSO:water
25:75. Compound 8c inhibited the assembly slightly compared
to the control. However, no Taxol-like effects were seen. Micro-
tubules disassembled rapidly upon lowering of the temperature
to 4 °C, and no decreased lag-phase or increase in assembly rate
was seen either if 8c was added initially or reassembled again
at 30 °C after disassembly at 4 °C.
The failure of 8c to stabilize microtubule is most likely due to
the lack of a benzoate group corresponding to the 2-benzoate
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 8 5 – 3 0 9 0
3 0 8 7

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in paclitaxel and also to the absence of the oxetane/4-OAc
grouping. Efforts to include these features will be reported in
due course.
at room temperature. 2-(2-Iodoethyl)-1,3-dioxolane (1.20 g,
6.14 mmol, 2.5 equiv.) was then added (the flask was covered
with aluminium foil) and the mixture was heated at 100 °C for
3 h whereafter ice was added together with a small amount of
water. The mixture was extracted with EtOAc (3 × 50 mL) and
then the combined organic phase was washed with brine (2 ×
15 mL), dried and the solvent was removed at reduced pressure.
The residue was purified by column chromatography (SiO2,
heptane:EtOAc 1:1) to give a mixture of the C- and O-alkylated
isomers (761 mg, 95%). Repeated chromatography gave the
following compounds in order of elution:
Experimental
Computations
Energy minimisations were initiated by running a full structure
energy minimisation using the Polak–Riber Conjugate gradient
(PRCG), (MM3*), followed by a Monte Carlo search (1000
cycles). Finally, a Full Matrix Newton Raphson (FMNR)
minimisation was performed with all the conformers found
within 1 kcal mol−1 from the global minimum in the Monte
Carlo search. To investigate the impact of the force field,
two minimisation series were made after the PRCG minimi-
sation, using two different force fields (MM3* and the Merck
Molecular Force Field (MMFF)). Since the core structure is
rather rigid, flexibility was only expected for the side chain and
the spiro cyclohexenone ring. As predicted, the obtained con-
formers from the two series showed a low degree of flexibility.
From the sequence using MM3*, six conformers were found
(DE = 0.64 kcal mol−1) with a total of five different orientations
of the formyl group and two for the spiro ring. For the MMFF
sequence, however, only four conformers were found (DE =
0.54 kcal mol−1), with two orientations of the formyl group and
two for the spiro ring. When comparing the lowest energy con-
former from each minimisation sequence, they showed different
orientations for both the formyl group and the spiro ring (see
Electronic Supplementary Information†). However, since the
energy difference between all the conformers in each sequence
was found to be very small, one can assume an equilibrium
between the different conformers. Therefore, when searching
for the structure with best resemblance with paclitaxel A, all
the obtained low energy conformers were analysed and 8a from
the MMFF sequence was found to be a good mimetic. No
solvent effects were taken into account in these methods, which
were implemented in the MacroModel™ version 7.1 molecular
modelling software (Schrödinger, Inc., Portland).26 The software
was executed on a Silicon Graphics CMNB024B model O2™
workstation (Silicon Graphics, Inc., Mountain View, CA).
13b. (75 mg, 9%); Rf 0.30; [a]D −103 (c 0.9 in CHCl3);
mmax(neat)/cm−1 2950, 2880, 1730, 1690, 1355, 1225; dH (CDCl3)
5.16 (1 H, ddd, J 9.3 Hz, 4.1 Hz and 2.0 Hz), 4.82 (1 H, t, J
4.1 Hz), 3.80–3.97 (4 H, m), 2.58 (1 H, m), 2.50 (1 H, m), 2.24
(3 H, s), 2.00–2.21 (3 H, m), 1.93 (3 H, s), 1.66–1.91 (5 H, m),
1.40–1.57 (2 H, m); dC (CDCl3) 213.23, 204.16, 169.61, 115.43,
103.43, 70.96, 67.08, 65.01, 65.00, 47.15, 31.73, 30.44, 27.71,
27.46, 27.03, 21.13, 20.89, 19.59; HRCIMS (CH4) Calcd. for
C17H25O6 325.1651. Found 325.1652.
13a. (360 mg, 45%); Rf 0.24; [a]D +52 (c 1.6 in CHCl3);
mmax(neat)/cm−1 2950, 2880, 1735, 1695, 1235; dH (CDCl3) 5.10
(1 H, dt, J 9.8 Hz, 3.2 Hz), 4.81 (1 H, t, J 4.3 Hz), 3.80–3.95
(4 H, m), 2.53 (1 H, m), 2.42 (1 H, m), 2.25 (3 H, s), 2.00–2.16
(2 H, m), 2.02 (3 H, s), 1.93 (1 H, m), 1.61–1.84 (5 H, m),
1.44–1.55 (2 H, m); dC (CDCl3) 212.09, 205.59, 170.07, 103.61,
71.80, 66.82, 64.96, 64.93, 47.54, 32.45, 30.44, 28.23, 27.75,
27.47, 21.56, 21.11, 18.95; HRCIMS (CH4) Calcd. for C17H25O6
325.1651. Found 325.1653.
The 2-O-alkylated major product was obtained from a fore-
run and a NOESY experiment (see below) indicated the posi-
tion of the alkylation; (95 mg, 12%); Rf 0.43; [a]D −21 (c 1.1 in
CHCl3); mmax(neat)/cm−1 2940, 2860, 1720, 1670, 1595, 1230; dH
(CDCl3) 5.04 (1 H, t, J 4.8 Hz), 4.98 (1 H, dt, J 8.7 Hz, 3.1 Hz),
4.11 (2 H, m), 3.85–3.99 (4 H, m), 3.46 (1 H, m), 3.26 (1 H, m),
2.40 (3 H, s), 2.11 (2 H, m), 2.01 (3 H, s), 1.99 (1 H, m), 1.63 (1
H, m), 1.50 (1 H, m), 1.38 (1 H, m), 1.19 (2 H, m); dC (CDCl3)
193.74, 170.58, 166.08, 119.88, 101.57, 73.22, 64.97, 64.91, 64.49,
35.26, 35.25, 34.14, 31.65, 27.75, 23.74, 22.09, 21.16; HRCIMS
(CH4) Calcd. for C17H25O6 325.1651. Found 325.1652. The two
O-alkylated products were taken together and converted back
to the starting material.
General
GC analyses were performed on a Varian 3400 gas chromato-
graph equipped with a DBwax (J&W Scientific) capillary
column (30 m, 0.25 mm i.d., 0.25 lm stationary phase). NMR
spectra were recorded on a Bruker DRX 400 spectrometer at
400 MHz (1H) and at 100 MHz (13C) using CDCl3 as solvent.
Signals from the solvent were used as reference lines (d 7.26 (1H)
and 77.0 (13C)) and J values are given in Hz. Infrared spectra
were recorded on a Shimadzu 8300 FTIR instrument. Optical
rotations were measured with a Perkin Elmer 241 polarimeter
at 21 °C at the sodium D-line. Chromatographic separations
were performed on Matrex Amicon normal phase silica gel 60
(0.035–0.070 mm) and for thin layer chromatography Merck
pre-coated TLC plates silica gel 60 F-254, 0.25 mm were used.
After elution the TLC plates were sprayed with a solution of
p-methoxybenzaldehyde (26 mL), glacial acetic acid (11 mL),
concentrated sulfuric acid (35 mL) and 95% ethanol (960 mL)
and the compounds were visualized upon heating. All solvents
were dried and distilled according to standard procedures,55
and the reactions were performed in septum-capped, oven-
dried flasks under an atmospheric pressure of argon. Organic
extracts were dried using Na2SO4 throughout. Mass spectra were
recorded on a JEOL JMDX 303 spectrometer.
The NOESY spectra of 13a,13b and the major O-alkylated
products were recorded using a mixing time of 800 ms. The
time domain data sets were accumulated over a sweep width of
1611 Hz using 2048 complex data points in the t2 dimension.
The repetition delay was 1.0 s and 64 scans were collected for
each t1 increment (256 in total). Total experimental time was
11 h 18 min.
(1R,3S,4S,6S)-Spiro[6-acetoxybicyclo[2.2.2]octan-2-one-3,1-
cyclohex-3-en]-2-one (14)
Compound 13a (200 mg, 0.62 mmol) was dissolved in 80%
acetic acid (25 mL) and heated at 65 °C for 3 h. The solution
was cooled to room temperature and then concentrated at
reduced pressure. In order to remove remaining water the
residue was co-evaporated three times with toluene under
reduced pressure. Neat TsOH (ca. 5 mg) was added to a
solution of the crude aldehyde in toluene (30 mL) and the
resulting mixture was heated at reflux temperature for 3 h in
a Soxhlet apparatus containing activated 4 Å molecular sieves
in the thimble. Half-saturated aqueous NaHCO3 was added at
0 °C, and the aqueous phase was extracted with EtOAc. The
combined organic solution was dried and then concentrated
at reduced pressure. The residue was chromatographed (SiO2,
heptane:EtOAc 1:1) to give 14 (138 mg, 85%) as a white solid;
mp 139–141 °C; [a]D −146 (c 0.3 in CHCl3); mmax(KBr)/cm−1
2960, 2935, 1730, 1660, 1235, 1030; dH (CDCl3) 6.89 (1 H, m),
(1R,3R,4S,6S)-3-Acetyl-3-(2-(2-oxolanyl)ethyl)-6-acetoxybi-
cyclo[2.2.2]octan-2-one (13a) and (1R,3S,4S,6S)-3-acetyl-3-(2-
(2-oxolanyl)ethyl)-6-acetoxybicyclo[2.2.2]octan-2-one (13b)
A solution of 1241 (554 mg, 2.47 mmol) in toluene (6 mL)
was added to Cs2CO3 (1.14 g, 3.50 mmol) in toluene (6 mL)
3 0 8 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 0 8 5 – 3 0 9 0

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5.95 (1 H, ddd, J 10.2, 2.6 and 1.6 Hz), 5.13 (1 H, dt, J 9.8 Hz,
3.4 Hz), 2.54 (1 H, m), 2.34–2.51 (3 H, m), 2.15–2.32 (3 H, m),
2.09 (1 H, m), 2.01 (3 H, s), 1.86 (1 H, dq, J 14.8, 3.1 Hz), 1.78
(1 H, m), 1.62 (1 H, m), 1.50 (1 H, m); dC (CDCl3) 211.79, 198.82,
170.06, 148.72, 128.58, 71.27, 61.77, 46.83, 31.30, 30.41, 26.93,
22.59, 21.13, 21.06, 18.47; HRCIMS (CH4) Calcd. for C15H19O4
263.1283. Found 263.1281.
126.83, 73.68, 73.06, 62.02, 54.46, 46.83, 30.66, 30.42, 26.96,
22.75, 20.97, 18.47; HRCIMS (CH4) Calcd. for C29H30O6N
488.2073. Found 488.2078.
Isolation of microtubule proteins and assembly studies
Cow brain microtubule proteins were isolated by two cycles of
assembly–disassembly as described in Wallin et al.56 Assembly
was measured at +30 °C. Microtubule proteins were drop-frozen
and kept in liquid nitrogen until used in experiments. Protein
concentrations were determined according to Lowry et al.,57
with bovine serum albumin as a standard.
(1R,3S,4S,6S)-Spiro[6-hydroxybicyclo[2.2.2]octan-2-one-3,1-
cyclohex-3-en]-2-one (15)
Solid K2CO3 (10 mg, 0.07 mmol) was added all at once to
a solution of 14 (100 mg, 0.38 mmol) in MeOH:H2O 9:1
(20 mL). The mixture was kept at room temperature for 2
h and then the solution was concentrated to approximately
4 mL at reduced pressure. The resulting solution was extracted
several times with EtOAc and the combined organic phase was
dried and concentrated at reduced pressure. The residue was
chromatographed (SiO2, heptane:EtOAc 1:4) to give 15 (72 mg,
86%) as a white solid; mp 135–136 °C; [a]D −43 (c 0.5 in CHCl3);
mmax(KBr)/cm−1 3020–3500, 2950, 2910, 1720, 1650, 1215; dH
(CDCl3) 6.88 (1 H, m), 5.93 (1 H, dt, J 10.2, 1.8 Hz), 4.23 (1 H,
dt, J 9.6 Hz, 3.0 Hz), 2.45 (1 H, m), 2.27–2.41 (4 H, m), 2.14
(1 H, ddd, J 14.8, 9.5 and 3.0 Hz), 2.05 (1 H, m), 1.88 (1 H, dq, J
14.7 Hz, 3.1 Hz), 1.79 (1 H, s), 1.56–1.73 (3 H, m), 1.45 (1 H, m);
dC (CDCl3) 213.51, 199.38, 148.87, 128.55, 69.60, 61.97, 51.13,
33.13, 30.58, 26.92, 22.69, 21.09, 18.43; HRCIMS (CH4) Calcd.
for C13H17O3 221.1178. Found 221.1174.
Assembly–disassembly of microtubules were measured in
a temperature-controlled spectrophotometer at 350 nm. The
assembly was induced by addition of 1 mM GTP and increasing
the temperature from 4 °C to 30 °C.
Acknowledgements
We thank the Swedish Science Council, The Crafoord Founda-
tion, The Royal Physiographic Society in Lund, The Research
School in Medicinal Sciences at Lund University and the Knut
and Alice Wallenberg Foundation for economic support. We
are grateful to professor Don Mastropaolo for sending us the
coordinates of the crystallographic structures of paclitaxel.
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