Synthesis and bioactivity of a side chain bridged paclitaxel: A test of the T-Taxol conformation

Article abstract of DOI:10.1016/j.bmcl.2009.03.063

A knowledge of the bioactive tubulin-binding conformation of paclitaxel (Taxol) is crucial to a full understanding of the bioactivity of this important anticancer drug, and potentially also to the design of simplified analogs. The bioactive con

Full text of DOI:10.1016/j.bmcl.2009.03.063

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2884–2887
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and bioactivity of a side chain bridged paclitaxel: A test of the
T-Taxol conformation
Mathis Hodge a, Qiao-Hong Chen a, Susan Bane b, Shubhada Sharma b, Maura Loew b, Abhijit Banerjee b,
Ana A. Alcaraz c, James P. Snyder c, David G. I. Kingston a,
*
a Department of Chemistry, M/C 0212, Virginia Polytechnic Institute and State University, 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, Atlanta, GA 30322, USA
a r t i c l e i n f o
a b s t r a c t
TM
Article history:
A knowledge of the bioactive tubulin-binding conformation of paclitaxel (Taxol ) is crucial to a full
understanding of the bioactivity of this important anticancer drug, and potentially also to the design
of simplified analogs. The bioactive conformation has been shown to be best approximated by the T-Taxol
conformation. As a further test of this conclusion, the paclitaxel analog 4 was designed as a compound
which has all the chemical functionality necessary for activity, but which cannot adopt the T-Taxol con-
formation. The synthesis and bioassay of 4 confirmed its lack of activity, and thus provided further sup-
port for the T-Taxol conformation as the bioactive tubulin-binding conformation.
Received 19 February 2009
Revised 10 March 2009
Accepted 10 March 2009
Available online 21 March 2009
Keywords:
Taxol
Paclitaxel
Ó 2009 Elsevier Ltd. All rights reserved.
Tubulin
Antiproliferative activity
T-Taxol
Conformation
Synthesis
The anticancer drug paclitaxel (1, TaxolTM, PTX) is one of the
most important anticancer natural products ever discovered, and
has contributed significantly to human health. First reported from
the Western yew, Taxus brevifolia, in 1971,1 it went through a long
period of development, and was finally approved by the FDA for
clinical use in 1992.
tubule-stabilizing drugs, the modified epothilone ixabepilone, has
recently been approved for clinical use.5
The normal functioning of tubulin assembly and disassembly is
crucial to cell division, and any interference with this process dis-
rupts cell division and leads to cell death by apoptosis. PTX owes
its activity to its ability to bind to tubulin and suppress microtu-
bule assembly dynamics.3 Thus the nature of the binding of paclit-
axel to tubulin, and in particular the tubulin-binding conformation
of PTX, is an important question with potential applications in the
design of improved taxoid drugs.
Since PTX has a large flexible side chain at C13 and smaller flex-
ible side chains at C2, C4, and C10, many different conformations
are possible. Various proposals for the binding conformation have
been made based on studies of the solution NMR spectra of PTX.
NMR studies in nonpolar solvents suggested a ‘nonpolar’ confor-
mation,6–8 while a ‘polar’ conformation featuring hydrophobic
interactions between the C2 benzoate, the C30 phenyl group and
the C4 acetate was proposed on the basis of NMR studies in polar
solvents.9–12 A third approach involved NMR studies using the
NAMFIS deconvolution method, and this showed that PTX adopts
9–10 conformations in CDCl3.13 An analysis of the electron crystal-
lographic data in combination with the NAMFIS results suggested
that the actual binding conformation has a T-shaped structure,
designated T-Taxol.14
AcO
O
OH
O
10
7
4
O
NH
Ph
2
2'
O
H
O
13
HO
OAc
OH
OBz
1
A turning point in its development came with the discovery of its
then unique mechanism of action as a promoter of the assembly
of tubulin into microtubules.2 The microtubule has been called
‘the single best cancer target identified to date’,3 and several other
microtubule-stabilizing natural products have been identified as
promising cancer therapeutics.4 The first of the non-taxane micro-
* Corresponding author.
E-mail address: dkingston@vt.edu (D.G.I. Kingston).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.063

M. Hodge et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2884–2887
2885
The validity of the T-Taxol model was demonstrated in several
ways. Direct evidence for the conformation was obtained by RE-
DOR NMR studies of labeled PTXs directly bound to tubulin. The
derived internuclear distances were compatible with the T-Taxol
tubulin-binding conformation, ruling out most competitors.15,16
The predictive value of this conformation was then tested by the
synthesis of bridged PTXs constrained by the bridging to the T-Tax-
ol conformation. The bridges were constructed between the C30-
phenyl group and the C4 acetate, since these two positions are
adjacent in the T-Taxol conformation. Two of the resulting bridged
compounds with two carbon chains connecting the ortho-position
on the C30-phenyl group to the acetate methyl group showed
excellent bioactivity, with activities 2–30-fold greater than those
of PTX, depending on the assay used.17,18
These results provided strong positive support for the T-Taxol
conformation as the bioactive conformation of PTX. It was also
desirable, however, to devise a negative test, to determine if the
T-Taxol conformation could also be used as a criterion for inactive
taxanes. Such a test is possible because of the extensive knowledge
of the structure–activity relationships of PTX.19–21
What the planar depiction does not show, however, is that com-
pound 4 cannot adopt the ‘T’ shape of T-Taxol represented by the
spatial arrangement of the three phenyl rings in 1.26 Thus, the
two well-separated aromatic rings at the termini of C13 have been
collapsed into a single bicycle occupying space between them as
shown in the manual docking of Figure 1a. This space is likewise
occupied by Val23 of the b-tubulin protein. If the T-Taxol geometry
or anything similar to it16,27 is required for bioactivity, then it is
predicted that 4 should be inactive, and such lack of activity would
be an important ‘negative test’ of these conformations.
The synthesis of 4 was accomplished by the Holton–Ojima b-
lactam synthon method28,29 from 10-deacetylbaccatin III, as shown
in Scheme 1. The key b-lactam 7 was prepared by a modification of
the standard conditions for b-lactam formation.30 In brief, 2-ben-
zyloxybenzaldehyde was converted to the b-lactam 6 by reaction
of its p-anisidine imine with acetoxyacetyl chloride in the presence
of Hünig’s base. Attempted resolution of 6 using Pseudomonas
cepacia lipase31 was unsuccessful, presumably because of the steric
hindrance from the ortho-benzyloxy group, and so the racemic
mixture was carried forward in the expectation that a kinetic res-
olution could be effected.32
Lactam 6 was hydrolyzed to the alcohol and reprotected as its
triisopropyl silyl derivative 7, which was then converted to the t-
butyloxycarbonyl derivative 8 by deprotection of the p-methoxy-
phenyl group with ceric ammonium nitrate and acylation with
di-tert-butyldicarbonate. b-Lactam 8 was then coupled with 7-(tri-
ethylsilyl)baccatin III33 under standard conditions to give the cou-
pled product 9. Hydrogenolysis of 9 followed by treatment with
formic acid gave the aminodiol 10, which was reacted with tri-
phosgene to give the cyclic carbamate 11. Deprotection of the 20-
TIPS group then gave the final product 4 (Scheme 1). The 1H
NMR spectrum of 4 and of its precursors 9–11 showed only one
set of signals for each of the protons, with no doubling of signals
due to the presence of two diastereomers.34 This indicated that
coupling occurred stereoselectively to give the desired product,
in agreement with the literature.32
The PTX analog docetaxel (2) and its 10-acetyl derivative 3 were
both prepared by Potier and his group from 10-deacetylbaccatin III,
and docetaxel has been developed into the second taxane for clin-
ical use.22 The 10-acetyl analog 3 is almost as active as docetaxel,
so the presence of the 10-acetyl group does not have a major effect
on activity.23
RO
O
OH
O
O
NH
Me3CO
O
H
OBz
O
HO
OAc
OH
2 R = H
3 R = Ac
The methyl carbamate 5a was prepared by standard methods.
The known non-racemic b-lactam 1235 was acylated with methyl
chloroformate to give the carbamate 13 (Scheme 2), and acylation
of 7-(triethylsilyl)baccatin III with 13 followed by deprotection
gave the analog 5a.
Constrained cyclic carbamate 4, open chain carbamate 5a, and
PTX were evaluated for tubulin-affinity, tubulin-assembly activ-
ity,36 and antiproliferative activity against the A2780 and PC3 cell
lines (Table 1). Association constants for PTX and 4 binding to GTP-
microtubules were assessed by a fluorescent competition assay as
described in the Supporting Information. Owing to limited solubil-
A simple modification of structure of 3 yields the target cyclic
carbamate 4, a compound with all the atom connectivity and struc-
tural features of PTX or docetaxel needed for bioactivity: an intact
tetracyclic taxane ring system, a C13 side chain with a C30-phenyl
ring and an N-acyl group, and a free C20-hydroxyl group. The
methyl carbamate 5a was selected as the best ‘open chain’ version
of 4 for comparison purposes, since it differs only by one carbon
atom from 4. Although the bioactivity of this compound has been
reported,24 we elected to synthesize it so as to allow it to be eval-
uated in the same assays used for the constrained compound 4. The
tolyl derivative 5b has been prepared previously,25 and was used
as a second comparison structure.
AcO
O
OH
O
O
O
NH
O
H
O
O
HO
4
AcO
OAc
OH
OBz
OH
O
O
R1
O
NH
O
H
OBz
HO
OAc
Figure 1. T-Taxol in b-tubulin (yellow): (a) best ROCS 3-D fit of T-Taxol and the
most similar T-conformer of compound 4 (blue) manually docked into b-tubulin.
Val23 on Helix 1 is in steric conflict with 4; (b) the best Glide docking of 4
(magenta) flips the ligand and directs the carbamate out into solvent.
OH
R2
5a R1 = OMe, R2 = H
5b R1 = Ph, R2 = Me

2886
M. Hodge et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2884–2887
OMe
OBn
OMe
OBn
NBoc
OBn
1. CAN, CH3CN,
1. KOH, THF,
0 oC, 61%
0 oC, 80%
2. TIPSCl,
Imidazole,
DMF, rt, 77%
N
N
2. (Boc)2O,
TIPSO
O
TIPSO
O
AcO
O
Et3N/CH2Cl2, 35
8
oC, 100%
7
6
1. 7-triethylsilyl baccatin III,
LHMDS, THF, - 50 oC, 78%
2. H2/Pd-C, rt, EtOAc, then
HCO2H, rt, 51% (2 steps)
AcO
O
AcO
OR3
OAc
O
OH
Triphosgene, py/CH2Cl2,
rt, 9 10, 62%
O
O
H
R2N
O
O
NH
O
O
H
OBz
O
O
HO
HO
OAc
OTIPS
OBz
OR
OR1
HF/py, THF,
11 R = TIPS
4 R = H
H2/Pd-C, Rt, EtOAc, then 9 R1 = PhCH2, R2 = Me3COCO, R3 = TES
0 oC - rt, 79%
HCO2H, rt, 51% (2 steps)
10 R1 = R2 = R3 = H
Scheme 1. Synthesis of cyclic carbamate 4.
erative activity as PTX in two different cell lines, being slightly
more active against the A2780 cell line and slightly less active
against the PC3 cell line. This result contrasts significantly with
the reported cytotoxicity data for compound 5a to P388 cells,
where it was 150-fold less active than docetaxel.24 The lack of
activity of 4 can thus be attributed, at least in large measure, to
the geometric constraint caused by the cyclic carbamate.
O
O
Methyl chloroformate
Et3N, DMAP, DCM
NH
N
OMe
65%
TIPSO
O
TIPSO
12
13
To place these results in a biostructural context, 4 was docked
flexibly into b-tubulin with Glide XP (See Supplementary data for
details).37 While the molecule can assume an overall shape similar
to the T-Taxol conformation, the cyclic carbamate moiety is re-
pelled by Val23-ligand congestion, rotates 4 ca. 180° relative to
Figure 1a and lifts it out of the binding pocket (Fig. 1b). Thus, the
best tolerated docking pose at the taxane site places 4 in a confor-
mation directing the carbamate ring out toward solvent. The con-
formation incorporates 9–10 kcal/mol internal strain energy
relative to the corresponding global minima using both MMFF
and OPLS2005 force fields.35 This estimate complements the obser-
vations by Buey et al.38 that taxane binding is enthalpy driven and
Scheme 2. Synthesis of b-lactam 13.
Table 1
Bioactivity of PTX and carbamates 4 and 5
Compd
Tubulin assembly
Tubulin affinity
Antiproliferative activity
(IC50, nM)
a
ED50
(
lM)
Kd (ꢀ107 Mꢁ1
)
A2780b
PC3b
PTX
4
5a
0.48 0.09c
>25
0.43 0.19
4.76 0.28
ꢂ0.03
14.9 4.0
>20,000
1.40 0.32
>5,000
2.00 0.08
1.11 0.04
9
1.9
a
that increasing binding enthalpy predicts cytotoxicity. The
DH con-
Disassociation constants (Kd) determined as described in Supplementary data.
Cytotoxicity determined as described in Supplementary data.
Data from Ref. 34.
b
c
tribution to G incorporates both intermolecular ligand–protein
D
interactions as well as intramolecular conformational strain en-
ergy. The calculation of substantial ligand torsional strain for 4
clearly counterbalances the otherwise favorable intermolecular en-
thalpy factor and aids in the interpretation of its low tubulin
assembly and antiproliferative activities.
Although these results do not prove that the T-Taxol shape is
the correct binding geometry, they do support the notion that a
taxane incapable of adopting a close mimic of the T-conformation
will not show significant tubulin-assembly activity, even though it
may possess all the correct chemical functionality for such activity.
Assessment of the alternative PTX-NY (‘REDOR-taxol’)26 conforma-
tion cannot be made, since this high energy conformer appears to
significantly reorganize the tubulin-taxane binding site, precluding
a simple comparison.39 Independent support for the T-Taxol con-
formation has been provided by a detailed analysis of REDOR
NMR data of labeled PTX analogs bound to microtubules.16
ity and low activity of compound 4, only a partial inhibition curve
could be obtained (Fig. 1 in Supplementary data). The association
constant measured for the binding of 4 to microtubules is therefore
an estimate based on limited data. However, the affinity of 4 for the
PTX site on microtubules is at least 150-fold less than that of PTX
(Table 1). The ability of 4 to promote in vitro tubulin assembly was
also evaluated. The efficacy of 4 in this assay is at least 50-fold less
than that of PTX. In addition, the cyclic carbamate 4 was unable to
stabilize microtubules against cold-induced disassembly (Figs. 2
and 3 in Supplementary data). Model compound 5a, on the other
hand, strongly promotes tubulin polymerization, with an activity
comparable to that of PTX. Model compound 5b is reported to have
an ED50 for tubulin assembly 5.1-fold greater than PTX, so the
introduction of an ortho substituent onto the 30-phenyl group
causes only a modest reduction in tubulin assembly activity.25
Cyclic carbamate 4 also exhibits 1900- to about 6000-fold less
antiproliferative activity than PTX in the two cell lines tested (Ta-
ble 1). Model compound 5a has approximately the same antiprolif-
Acknowledgements
This work was supported by NIH Grant CA-69571; we are grate-
ful for this support. We thank William Bebout (VPISU) for mass

M. Hodge et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2884–2887
2887
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Supplementary data
Supplementary data (experimental procedures for the synthesis
of all compounds, for the molecular modeling, and for the tubulin
bioassays) provided as supplementary material associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2009.03.063.
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