Evaluation of the tubulin-bound paclitaxel conformation: Synthesis, biology, and SAR studies of C-4 to C-3′ bridged paclitaxel analogues

Article abstract of DOI:10.1021/jm061071x

The important anticancer drug paclitaxel binds to the β-subunit of the ββ-tubulin dimer in the microtubule in a stoichiometric ratio, promoting microtubule polymerization and stability. The conformation of microtubule-bound drug has been the subject of in

Full text of DOI:10.1021/jm061071x

J. Med. Chem. 2007, 50, 713-725
713
Evaluation of the Tubulin-Bound Paclitaxel Conformation: Synthesis, Biology, and SAR
Studies of C-4 to C-3′ Bridged Paclitaxel Analogues
Thota Ganesh,† Chao Yang,† Andrew Norris,† Tom Glass,† Susan Bane,‡,* Rudravajhala Ravindra,‡ Abhijit Banerjee,‡
Belhu Metaferia,† Shala L. Thomas, Paraskevi Giannakakou, Ana A. Alcaraz,§ Ami S. Lakdawala,§ James P. Snyder,§,* and
David G. I. Kingston†,*
Department of Chemistry, M/C 0212, Virginia Polytechnic Institute and State UniVersity, Blacksburg, Virginia 24061, Department of
Chemistry, State UniVersity of New York at Binghamton, Binghamton, New York 13902, Winship Cancer Institute, Emory UniVersity School of
Medicine, Atlanta, Georgia 30322, and Department of Chemistry, Emory UniVersity, Atlanta, Georgia 30322
ReceiVed September 11, 2006
The important anticancer drug paclitaxel binds to the â-subunit of the Râ-tubulin dimer in the microtubule
in a stoichiometric ratio, promoting microtubule polymerization and stability. The conformation of
microtubule-bound drug has been the subject of intense study, and various suggestions have been proposed.
In previous work we presented experimental and theoretical evidence that paclitaxel adopts a T-shaped
conformation when it is bound to tubulin. In this study we report additional experimental data and calculations
that delineate the allowable parameters for effective paclitaxel-tubulin interactions.
Introduction
The nature of the binding of PTX to tubulin polymer is not
yet known in complete detail, although a significant advance
was made by the determination of the electron crystallographic
structure of R,â-tubulin, initially at 6.5 Å and later improved
to 3.7 Å.12,13 Although the latter work was carried out with PTX-
stabilized zinc-induced sheets, it lacks the resolution to define
the detailed conformation of paclitaxel on the tubulin polymer.
PTX is a large and conformationally mobile molecule, with
rotatable groups at C-2, C-4, C-10, and C-13 appended to an
essentially rigid tetracyclic ring system. The question of the
preferred binding conformation of PTX on the binding site of
the tubulin receptor is thus a complicated one.
The natural product paclitaxel (PTX)a (1a) and its closely
related semisynthetic analogue docetaxel (DTX, 1b) are clini-
cally approved drugs for several tumor malignancies, including
breast, lung, and ovarian carcinomas,1 while PTX has recently
been shown to be effective in reducing the risk of restenosis
following percutaneous coronary intervention.2 These molecules,
in common with several other recently discovered natural
products such as the epothilones,3 discodermolide,4 laulimalide,5
and eleutherobin,6 act by promoting the polymerization of
tubulin to stabilized microtubules, leading to cell cycle arrest
at the G2/M phase and hence to apoptotic cell death.7-10
Although PTX has been shown to influence the phosphorylation
and translocation of cell signaling factors,11 its clinical activity
is believed to be directly related to its microtubule-binding
activity.10
Previous studies of the bioactive conformation have used
evidence from NMR and modeling analyses to suggest three
different models for the bioactive conformation of PTX. A
“nonpolar” conformation with clustering of the C-2 benzoate
and the C-3′ benzamido group of the side chain was proposed
as the bioactive conformer on the basis of single conformation
NMR studies in nonpolar solvents.14-16 Similar NMR studies
in polar solvents revealed a hydrophobically collapsed confor-
mation with clustering of the C-2 benzoate and the C-3′ phenyl
group. This led to the proposal that the “polar” conformation is
the bioactive form.17-21 Although most of the reports assumed
22
a single conformation, deconvolution for PTX in CDCl3 and
D2O/DMSO-d623 makes it clear that the molecule adopts 8-14
conformations, no one of which achieves a population above
30%. A second approach has used spectroscopic studies of
tubulin-bound PTX. One study using REDOR NMR provided
F-13C distances of 9.8 and 10.3 Å between the fluorine of a
2-(p-fluorobenzoyl)PTX and the C-3′ amide carbonyl and C-3′
methine carbons, respectively.24 A related solid-state study
employing the NMR RFDR method reported a distance of 5.5
Å between the fluorines of 2-(p-fluorobenzoyl)-3′-(p-fluorophen-
yl)-10-acetyl-DTX.21 Both of the latter investigations singled
out the polar conformation of PTX as the bound rotamer.
The “polar” and “nonpolar” conformations have inspired a
number of elegant synthetic studies designed to generate
constrained analogues which maintain these conformations.
However, with one exception,25 none of the constrained
analogues synthesized to date based on these conformations have
yielded analogues with both tubulin polymerization and cyto-
PTX is a complex molecule and is expensive to produce from
natural sources. Ideally, future generations of this class of drugs
should have much simpler structures, while retaining the activity
of the parent drug. Any rational design of improved or simplified
analogues would be greatly enhanced by knowledge of the
“pharmacophore” of PTX, which can be approximately equated
with the conformation of this compound in its binding site on
â-tubulin. Thus the elucidation of this pharmacophore is a matter
of great practical as well as theoretical significance.
* To whom correspondence should be addressed. Phone: 540-231-6570.
Fax: 540-231-3255. E-mail: dkingston@vt.edu.
† Virginia Polytechnic Institute and State University.
‡ State University of New York at Binghamton.
§ Department of Chemistry, Emory University.
Emory University School of Medicine.
a Abbreviations: PTX, paclitaxel; NAMFIS, NMR analysis of molecular
flexibility in solution.
10.1021/jm061071x CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/31/2007

714 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Ganesh et al.
toxic activities equal to or greater than those of PTX itself. Thus,
various compounds designed to mimic the polar conformation
have been prepared, but these compounds were either inactive26
or less active than PTX.27 Similarly, constrained analogues based
on the nonpolar conformation were also less active than
PTX.28-31 Two recent studies have presented two bridged
taxanes with microtubule stabilizing capacity equivalent to
paclitaxel, but with diminished cytotoxicity.32 In a review of
taxane-bridging strategies, we have detailed the need for a new
approach and outlined our initial efforts in this direction.33
Results and Discussion
Figure 1. T-Taxol illustrating the short H-H distance between the
centroid of the C-4 acetate methyl group and the ortho-position of the
C-3′ phenyl ring.
Design and Development of T-Taxol Models. As outlined
above, conception of various bridged analogues as candidates
for binding at the taxane site on â-tubulin followed from
hypotheses regarding the nature of the binding pose. In this
respect, the T-Taxol conformation is no exception. The ground-
breaking work of Nogales, Wolf, and Downing that solved the
structure of Râ-tubulin by electron crystallography (EC) was
made possible, in part, by stabilizing sheets of polymerized
tubulin with zinc cations and PTX.12 The 3.7 Å resolution
structure, however, was unable to reveal paclitaxel’s conforma-
tion and binding pose. The molecule’s location was nonetheless
represented by the single-crystal X-ray structure of docetaxel
introduced as a ligand place-holder.34 To overcome the paucity
of structural detail for the ligand, a strategy combining the low-
resolution EC data and higher resolution small molecule
structural data was employed.
lecular modeling for the T-Taxol model,39 and we have also
shown that the model can guide the synthesis of cytotoxic
analogues from non-cytotoxic precursors.40 In this paper, we
describe the synthesis, biological evaluation, and molecular
modeling for an expanded set of highly active C-4 to C-3′
constrained analogues.
Synthesis of Macrocyclic PTX analogues. We selected the
well-established ring-closing metathesis strategy41 for the crucial
macrocyclization step (Scheme 1), as it has proved highly
efficacious for generation of macrocyclic taxoids.27 The pro-
posed macrocyclic analogues (A) were envisioned to arise from
the open chain ω,ω′-dienes B, which can be derived from
modified â-lactam (C) and baccatin III (D) derivatives.
The retrosynthesis shown in Scheme 1 required the prepara-
tion of several â-lactam derivatives with a substituent at either
the ortho or meta position of the phenyl group and various C-4
modified baccatin derivatives. The syntheses of the â-lactam
derivatives were accomplished by application of literature
procedures42,43 (Scheme 2). The syntheses of (()-â-lactams
(3a-e) were achieved starting from 3-allyloxy, 3-vinyl, 2-al-
lyloxy, 2-butenyloxy, and 2-bromo benzaldehydes (1a-e)
through the N-(p-methoxy phenyl) (PMP) protected imines 2a-
e. Resolution of the (()-â-lactams (3a-e) was carried out with
lipase PS (Amano) to yield the desired enantiomeric acetates
(+)-(4a-e), along with undesired enantiomeric (-)-alcohols
(not shown) in more than 95% yield.43,44 Functional group
manipulations on 4a-e generated the triisopropylsilyl ether
intermediates 5a-e. The 4-(2-bromophenyl) derivative (5e) on
Stille coupling45 with vinyltributyltin and allyltributyltin pro-
duced the 4-(2-vinylphenyl) and 4-(2-allylphenyl) lactam in-
termediates 5f,g, respectively. The PMP-protected intermediates
5a-d and 5f,g were deprotected with ceric ammonium nitrate
to produce secondary amides (6a-d,f,g), which were treated
with benzoyl chloride in the presence of triethylamine and
dimethylaminopyridine to result in the (+)-(3R,4S)-1-benzoyl-
3-triisopropylsilyloxy-4-(aryl)-azetidin-2-ones 7a-d and 7f,g.
(Scheme 2)
Synthesis of the baccatin derivatives 11a-d started with the
known 4-deacetylbaccatin derivative 8.46 Initial attempts to
acylate the hindered C-4 hydroxy group using the DCC/DMAP
method resulted in unacceptable yields of C-4 acyl derivatives,
but the use of acid chloride and LiHMDS in THF at 0 °C gave
the C-4 acyl baccatin derivatives 9a-d with yields ranging from
50 to 78%. A minor product acylated at the C-4 position, but
with a loss of the 1-dimethylsilyl group, was always obtained
in this step, and it was used for the subsequent step. Global
deprotection of 9a-d using HF·pyridine, followed by selective
C-10 acetylation47 with 0.1 mol % of CeCl3 and acetic anhydride
in tetrahydrofuran, gave greater than 90% yields of the desired
10-acetyl derivatives (10a-d). Selective protection of the C-7
Specifically, an analysis of the 2D NMR spectra of PTX in
22
23
both CDCl3 and D2O/DMSO-d4 using NAMFIS methodol-
ogy35 indicated that the compound adopts over a dozen
conformations, among which a T-Taxol or butterfly conforma-
tion contributes mole fractions of 0.04 (4% population) and 0.02
(2%), respectively, in the two solvents. EC-density fitting of
both the NMR conformations and a number of taxane X-ray
structures led to the postulate that the T-Taxol conformation is
the bioactive form.36 This model posits that the centroid of the
C-2 benzoate phenyl of PTX is essentially equidistant from the
centroids of the phenyl and benzamide-phenyl groups emanating
from the C-3′ position; thus, the T-shaped conformation. A
singular advantage of this structural motif is that when the
T-Taxol conformer is nestled into the electron crystallographic
density of the PTX-tubulin complex, His227 of the protein is
interposed between the C-3′ benzamido and C-2 benzoyl phenyl
rings of PTX. Hydrophobic collapse of the taxane phenyl groups
in this sector of the binding pocket is thereby eliminated as a
feature of ligand binding.36
Examination of the T-shape shows that the C-3′ phenyl and
C-4 OAc moieties are juxtaposed, the distance between the
o-phenyl and methyl centroid hydrogens being only 2.5 Å
(Figure 1).37 If the T-Taxol model accurately reflects the PTX-
tubulin interaction, conformationally constrained analogues
which maintain this juxtaposition should yield PTX analogues
with improved tubulin-binding properties. The entropic penalty
resulting from binding to tubulin is greatly reduced in the
constrained analogues. As a result these analogues were expected
to show a higher bioactivity than PTX itself, particularly when
the bridge is short and emanates from the C-3′ phenyl ortho
center. The predictions were vindicated by our preliminary
results in this area, which demonstrated that taxoid design and
synthesis based on the T-Taxol conformation yields compounds
with improved activity in both tubulin polymerization and
cytotoxicity assays.37,38 Very recently we have presented ad-
ditional experimental evidence from REDOR NMR and mo-

Synthesis and SAR Studies of the BioactiVe Paclitaxel Conformation
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 715
Scheme 1. Retrosynthetic Logic for Preparation of Macrocyclic Taxanes A from ω,ω′-Dienes B Derived from â-Lactams C and
Baccatins III D
Scheme 2. Synthesis of â-Lactams 7a
a a. p-MeOC6H4NH2, MgSO4, CH2Cl2 (100%); b. CH3COOCH2COCl, Et3N or Pri2EtN, -78 °C to room temp, 12 h (80-85%); c. lipase PS (Amano),
phosphate buffer, pH ) 7.2, CH3CN, 24 h to 12d (90-95%) d. i, 1 M, KOH, THF, 0 °C (quantitative); ii, TIPSCl, imidazole, DMF (90-94%); e. CAN,
CH3CN, 0 °C (65-92%). f. Pd2(dba)3, Ph3P, dioxane, 80 °C, vinyltributyltin (5f, 80%), allyltributyltin (5g, 84%). g. (i) CAN, CH3CN, -5 °C (65-70%).
(ii) PhCOCl, Et3N, DMAP, CH2Cl2 (7f, 86%, and 7g, 90%); h. PhCOCl, Et3N, DMAP, CH2Cl2 (85-95%).
Scheme 3. Synthesis of Baccatins 11a-da
a a. LHMDS, THF, 0 °C, acryloyl chloride (9a, 52%), 4-pentenoyl chloride (9b, 78%), 5-hexenoyl chloride (9c, 71%), 6-heptenoyl chloride (9d, 50%);
b. i, HF·Py, THF (n ) 0, 60%, n ) 2, 91%, n ) 3, 80%, n ) 4, 44%). ii, CeCl3, Ac2O, THF (10a, n ) 0, 89%), (10b, n ) 2, 92%), (10c, n ) 3, 89%),
(10d, n ) 4, 95%); c. Triethylsilyl chloride, imidazole, DMF (11a, n ) 0, 87%), (11b, n ) 2, 85%), (11c, n ) 3, 72%), (11d, n ) 83%).
hydroxyl as its triethylsilyl ether afforded the C-4 alkenoyl
baccatins 11a-d in good yields (Scheme 3).
E forms of the bridging double bond were obtained, but for
reasons we do not fully understand, only the Z form of
compound 15f was obtained. The Z configuration was assigned
on the basis of the coupling constant of 11.4 Hz for the olefinic
protons of the bridging double bond. It is noteworthy that
compound 23, with a m-methoxybenzoyl group at C-2, gave a
mixture of the Z-alkene 27 and the unconjugated E-alkene 24
on olefin metathesis (see below).
Having prepared the desired baccatin III derivatives (11a-
d) and â-lactam derivatives (7a-d,f,g), we synthesized the ω,ω′-
diene taxoid precursors 12a-k, with yields ranging from 50 to
92% using the Holton-Ojima coupling protocols48 (Scheme 4).
These dienes set the stage for the crucial ring-closing metathesis
reaction.
Our initial studies of the cyclization reaction employed the
ω,ω′-diene substrates 12g and 12h and first generation Grubb’s
catalyst in dichloromethane under high dilution condition,38 but
in later studies the second generation Grubbs’s catalyst under
the same high dilution conditions proved to give superior results.
In this particular transformation the open chain diene substrates
12f-h produce exclusively the Z alkenes 14f-h, while the
dienes 12a and 12c gave exclusively the E-alkenes 14a and 14c.
The remaining dienes 12d and 12i-k yielded E/Z mixtures 14d,
14i-k which were separable by silica gel chromatography. All
the cyclic derivatives 14 were subjected to deprotection with
HF·Py in tetrahydrofuran to produce the bridged paclitaxel
analogues 15 in satisfactory yields. In most cases the more stable
In a preliminary effort to explore the absence of E-15f from
the reactions just described, we carried out density functional
theory (DFT) single point calculations on MMFFs E and Z
optimized structures (model: B3LYP/6-31G**). Indeed, in
contrast to the experimental outcome, the E-form is predicted
to be more stable than the Z-isomer. Preliminary DFT optimiza-
tion of truncated forms of Z-15f and E-15f provided a similar
relative energy. We speculate that the outcome of ring closure
metathesis for 15f is not governed by product thermodynamics,
but by the geometry of the bulky Grubbs catalyst in complex
with the starting diene or the corresponding transition state.
Finally the saturated bridged paclitaxels 16 were prepared
by hydrogenation of 15 (Scheme 5). It is worth noting here that

716 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Scheme 4. Synthesis of Bis-Alkenyl Baccatins 13a-ja
Ganesh et al.
a a. LHMDS, THF, -40 °C; b. NaH, THF, 0 °C to room temp; c. HF·Py in THF, 0 °C to room temp, 12 h.
neither the dienes 12b and 12e nor their 2′,7-silyl deprotected
derivatives 13b and 13e yielded any of the expected olefin
metathesis products 14b and 14e under any of the several ring-
closing metathesis conditions tested using either Grubbs cata-
lysts, presumably due to the ring stain inherent in forming the
short bridges required for these compounds.
16i,j with 8-10 atoms in the bridge, respectively. These
compounds were approximately 10-130 fold less active than
PTX in both cell lines. Interestingly, and in contrast to 15g and
15h, the open chain analogues 13i,j were found to be inactive
or only weakly active against the A2780 cancer cell line (Table
1).
Development of Bridged PTX Analogues with Improved
Bioactivities. As pointed out in the introduction, a key test of
the proposed bioactive conformations is the synthesis of a
constrained analogue that mimics this conformation and pos-
sesses an equal or greater bioactivity with respect to the parent
compound. In our approach to the synthesis of such a confor-
mationally constrained analogue, we initially synthesized the
21- and 19-membered macrocyclic analogues 15g and 15h with
eight and six atoms, respectively, in a bridge between the C-4
acetyl group and the meta-position of the C-3′ phenyl group of
PTX.38 These analogues exhibited modest cytotoxicity against
the A2780 ovarian cancer cell line and significant tubulin
polymerization (TP) activity, but the activities were considerably
less than those of PTX. The reduced activity of 15g was
explained by a combination of NMR-NAMFIS analysis, which
deconvolutes NMR virtual structures to individual conformers
in solution, and a computational analysis of taxane-tubulin
complexes. These studies indicated that compound 15g, although
capable of adopting the bioactive T-form, is seated higher in
the PTX-binding pocket as a result of close contact between
the propene moiety of the tether and Phe270 of the protein.38,49
In the present study, we initially synthesized the dihydro
derivatives 16g and 16h by hydrogenation of 15g and 15h,
respectively, to evaluate whether increased flexibility of alkane-
bridged macrocyclic taxoids might alleviate the unfavorable
interaction with Phe270.49 However, the bioactivities of these
compounds with saturated bridges were even less than those of
the unsaturated compounds 15g and 15h. Surprisingly, the open
chain analogues 13g and 13h showed better cytotoxicities than
their macrocyclic counterparts 15g and 15h (Table 1).
At this point, we investigated the synthesis of compounds
with shorter bridges. Thus, the two macrocyclic taxoids 15c
and 15d and their saturated dihydro derivatives 16c and 16d,
with six or seven atoms in the bridge between C-4 and the C-3′
o-phenyl position of PTX were prepared. The compounds show
approximately equal cytotoxicity to PTX against the A2780
ovarian cancer cell line but reduced activity against the PC3
prostate cancer cell line. Comprehensive conformational NMR/
NAMFIS and docking studies indicated that compound 15c was
the best fit for the T-conformation of PTX. By modeling, this
compound not only seats itself into the tubulin binding pocket,
escaping the steric clash observed for meta-bridged compounds
15g and 15h,37,38 but it also nicely accommodates His227 of
â-tubulin, allowing the imidazole ring to insert itself between
the stacked rings (cf. Figure 2).37
We sought to refine the bridge in 15c further by shrinking
the number of connecting atoms. Consequently the 17-
membered macrocyclic taxoid 15f and its saturated dihydro
derivative 16f, with 7 atoms in the bridge linking the ortho
position of the C-3′ phenyl to the C-4 position, were synthesized.
Gratifyingly, the bridged taxoid 15f exhibited excellent bio-
activity. It is at least 50 times more potent than PTX against
A2780, while its dihydro derivative 16f shows about 30 times
more activity than PTX against the same cell line. Both bridged
taxoids also show slightly increased cytotoxicity compared with
PTX against the PC3 cell line. The unusual activities of 15c,d,
15f, and 16c,d, 16f are not due to the substituents on the C-3′
phenyl or the C-4 positions, since the open chain analogues
13c,d and 13f are almost completely inactive or weakly active
against both the A2780 and PC3 cell lines (Table 1). This
indicates that the activity associated with the bridged taxoids
must originate from their conformational restriction.
Molecular modeling studies of the interaction of macrocyclic
taxoids linked from the ortho position of the C-3′ phenyl group
to the C-4 position indicated that these compounds are ideally
suited to maintain the T-Taxol conformation while avoiding the
unfavorable interaction with Phe270. Thus, these compounds
were targeted as especially attractive candidates for synthesis.
We adopted a similar ring-closing metathesis strategy to that
used for 15g and 15h and subsequently synthesized the ortho-
linked macrocyclic taxoids 15i-k and their dihydro derivatives
Molecular modeling studies suggested that the E bridged
macrocyclic analogue of 15f should be more stable than Z-15f,
while minireceptor QSAR50 predicts this compound to be more
active than PTX. On the basis of these projections, we
investigated synthesis of the E macrocyclic bridged derivative
of 15f. Subjection of diene 12f to ring-closing metathesis with
Grubbs second generation catalyst in dichloromethane at an

Synthesis and SAR Studies of the BioactiVe Paclitaxel Conformation
Scheme 5. Synthesis of Bridged Paclitaxels 15 and 16a
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 717
a a. (H2IMes)(PCy3)(Cl)2Ru)CHPh, CH2Cl2, room temp; b. (PCy3)2(Cl2)Ru)CHPh, DCM, room temp; c. HF, Py, THF, 0 °C to room temp d. H2, Pd/C,
35 psi.
elevated temperature of 55 °C produced exclusively the isomer-
Synthesis of Bridged Paclitaxel Derivatives with Modified
Pendent Groups. Having optimized the macrocyclic bridge
between the C-4 acyl and the C-3′ o-phenyl positions, we next
investigated the structure-activity relationships at other sites
of the bridged paclitaxel analogues in an attempt to identify
additional paclitaxel analogues with further improved activity.
We51 and others52 have previously reported the unusual cyto-
toxicity and in Vitro tubulin polymerization activity of various
ized product 17f, which on deprotection with HF·Py furnished
the isomerized E alkene 18f (Scheme 6). This compound showed
excellent cytotoxicity, almost equal to that of the Z bridged
derivative 15f, indicating that the precise stereochemistry of the
bridging alkene linker causes only a small difference in the
activity. The structure of 18f was confirmed by hydrogenation
to give a dihydro derivative identical with 16f.

718 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Ganesh et al.
Table 1. Cytotoxicity and Tubulin Polymerization Activity of
Macrocyclic and Open Chain Paclitaxel Analogues
macrocyclic paclitaxel analogue 28. It was accomplished using
modifications of known reactions. Thus 7,10,13-tris(triethyl-
silyl)-10-deacetylbaccatin (III) 19 was debenzoylated with Red-
Al followed by protection as its cyclic carbonate 20.27 Com-
pound 20 was treated with m-methoxyphenyllithium at 0 °C,
followed by dimethylsilyl chloride to give 21 in 70% overall
yield. Finally 21 was converted to the key intermediate 22 by
steps similar to those of Scheme 2. Coupling of the building
blocks 22 and 7g under standard conditions48 furnished the diene
23 in 70% yield. Ring-closing metathesis of diene 23 using
second generation Grubbs’s catalyst yielded the bridged taxoids
24 and 27 in a 3:1 ratio, with no formation of E-alkene.
Interestingly, the first generation Grubbs’s catalyst failed to bring
about macrocyclization under various solvent and temperature
conditions. The final compounds 25 and 28 were obtained by
deprotection with HF·Py in tetrahydrofuran (Scheme 7), while
hydrogenation of the two isomers delivered the same saturated
compound 26. Disappointingly, the macrocyclic bridged m-
methoxy derivative Z-28 and its isomer 25, both showed reduced
activity against the A2780 and PC3 cell lines compared with
the corresponding C-2 benzoyl derivative Z-15f, and the dihydro
derivative 26 was also less cytotoxic to the A2780 cell line (it
was not tested against the PC3 cell line). The N-Boc derivatives
31a and 32b were however more promising, and in this series
the C-2 m-methoxybenzoyl group did not reduce activity
significantly in compounds 31a, 32a, and 32b, in contrast to
its effect on the N-benzoyl series. The reasons for this difference
between the effects of the C-2 m-methoxybenzoyl group on the
two different series are not clear. It is noteworthy that
compounds 32a and 32b were the most potent compounds of
the entire series against the PC3 cell line and also as promoters
of tubulin polymerization.
IC50 values (nM)a,b
tubulin polym
compound
A2780
PC3
(ED50, µM)b,g
PTX
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
15a
15c
15d
15f
15g
15h
15i
15c
1900
1700
17800
19800
1600c
1300e
770c
350
5
550
320
0.5
1.0
1.0
2800c
>13200
275
1.2c
2.7c
1.4
550
128
55
2600
4600c
580
15
16
1.4
0.42
0.49
1.8
2520
7400
6300
14.5
20d
2.2
4.6c
0.28
0.67
0.3
0.3d
650
680
830
2.5
NDf
34
NDf
1.6
55
0.9
15j
15k
16a
16c
16d
16f
16g
16h
16i
16j
18f
25
26
28
440
90
0.53
0.93
1.4
0.83
0.23
0.21
3.4c
1.4
0.76
0.85
0.33
0.57h
NDf
0.22h
0.49h
0.35h
0.18h
0.22h
1840c
700d
18.5d
28
500
53
50c
13
0.5d
2.4
3600c
8800
980c
2000
0.5d
1000
570
51
390c
3.1
23.7d
23.1e
17.7d
1.4d
6
NDf
8.2
3.2
11
1.5
1.3
31a
31b
32a
32b
>6i
0.65d
0.49d
We then elected to carry out structural modifications on the
side chain of the bridged paclitaxel analogues, since docetaxel
with an N-Boc substituent has improved activity as compared
with PTX. The â-lactam derivative 7h was prepared by the
procedure described Scheme 2. Coupling of 7h individually with
11a and 20 gave ω,ω’-dienes 29a and 29b, and these dienes
upon ring-closing metathesis produced exclusively the cyclic
double bond isomerized products 30a and 30b. None of the
normal Z or E unisomerized alkenes were obtained in this
reaction. The usual HF·Py deprotection produced the products
31a and 31b, and hydrogenation furnished the dihydro deriva-
tives 32a and 32b (Scheme 8).
a Average of three determinations unless otherwise stated. b Standard error
is less than 10% of the mean unless otherwise stated. c Standard error
between 10% and 25% of the mean. d Standard error greater than 25% of
the mean. e Single determination. f ND: Samples were not tested. g ED50
values determined with GTP-tubulin unless otherwise stated. h ED50 values
for these samples were determined with GDP-tubulin and were normalized
to the GTP-tubulin scale. i The value cited is the lower of two widely
different determinations, probably due to solubility problems.
The bioactivities of compounds 31a, 32a and 32b were 10-
30-fold greater than those of PTX in the A2780 cell line, while
in the PC3 cell line these compounds were 1.5 to 4-fold more
potent than PTX, and 31b was about half as potent as PTX in
this cell line. Since the corresponding PTX analogues 15f and
16f are 30-50-fold more potent than PTX against the A2780
cell line and twice as potent as PTX against the PC3 cell line,
it appears that the additional activities generally conferred by
the N-Boc group and the m-methoxybenzoyl group are unique
to the unbridged compounds. Modeling suggests a modest
reorientation of the terminal phenyl rings in bridged compounds
within the taxane binding site.53 While the associated binding
pose contributes to improved bridge-induced tubulin binding,
it apparently dampens these additive substitutent effects at the
same time.
Figure 2. T-Conformations of PTX (blue) and 15f (red) in the â-tubulin
binding site, the latter having been docked by the Glide software. Both
conformations were derived by NAMFIS analyses. His227 is shown
in front of the structures, the 15f bridge behind, and Phe270 (ref 49)
further behind.
Historically, the search for microtubule modifiers has often
been accompanied by a preliminary assessment of biological
activity by examination of a drug’s ability to polymerize tubulin
or to influence the stability of microtubules. More recently, this
practice has been largely supplanted by a focus on cytotoxicity.
C-2 aroyl-substituted paclitaxel analogues, such as the C-2
m-methoxybenzoyl and m-azidobenzoyl derivatives. We thus
embarked on the synthesis of the C-2 m-methoxybenzoyl

Synthesis and SAR Studies of the BioactiVe Paclitaxel Conformation
Scheme 6. Synthesis of Bridged Paclitaxel 18a
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 719
a a. Grubbs catalyst, CH2Cl2, 55 °C, 77%; b. HF/Py, THF, 77%.
Scheme 7. Synthesis of Bridged Paclitaxels 25-28a
a a. i, Red-Al, THF, 0 °C, ii, carbonyldiimidazole, imidazole, 33% (overal two steps); b. i, m-methoxyphenyl-MgBr, Et2O, 0 °C, ii, DMSCl, imidazole,
70% (oveal two steps); c. i, Red-Al, THF, 0 °C, 55%, ii, LHMDS, CH2)CHCOCl, THF, 43%, iii, HF/Py, THF, 0 °C to room temp, 70%, iv, Ac2O, CeCl3,
THF, room temp, 90%, v, TESCl, imidazole, CH2Cl2, 84%; d. LHMDS, THF, 90%; e. Grubbs catalyst (2nd), CH2Cl2, room temp, 70%; f. HF/Py, THF,
80%; g. H2, Pd/C, MeOH, 60%.
The main reason, of course, is that an effective drug needs to
penetrate the membrane of a cell to arrest microtubule function
within its boundaries. Factors such as cell permeability,
cytoplasmic metabolism, and nonselective binding are, of course,
unexamined by an in Vitro appraisal of the state of microtubule
polymerization. As a consequence, in many cases where both
polymerization and cytotoxicity data are available, a correlation
fails to emerge. This is the case in the present study. For
example, numerous analogues are equal to or within a factor of
2 relative to PTX in the polymerization assay, but 20-1200
fold poorer in the cell-based assays (e.g., 13a,b,c,g,h, 15i,j, and
16i,j). By the same token, other in Vitro analogues whose
activities differ by a factor of 2 from PTX (15c,d, 25, 28, 31a,
32b) are nearly equivalent to PTX in their cellular action.
Measurements of critical concentration and Kp, the equilibrium
constant for polymer growth, would appear to be a much more
reliable approach to assessment of biological activity.37 Such
measurements will be the subject of future work by our
laboratories.
these clones revealed that the resistance phenotype was due to
distinct acquired â-tubulin mutations at the taxane binding
pocket.54,55 As a result, PTX’s ability to interact with the mutant
tubulins in these clones is significantly impaired as evidenced
by the lack of drug-induced microtubule-stabilizing activity and
essentially lack of G2/M arrest.56 Since drug resistance is the
factor that hampers most PTX’s clinical activity, we set out to
test the activity of our analogues against the parental and drug-
resistant cell lines (Table 2). While 13g and 15c exhibited similar
or slightly improved activities over PTX, there are two
compounds that stand out. Specifically the bridged taxoid 15f
and the dihydro analogue 16f exhibit a remarkable activity in
that not only are they about 100-fold more active than PTX
against the parental 1A9 cells, but they are also 1200- and 150-
times more effective than PTX (respectively) toward the PTX-
resistant cell line PTX10 (Table 2). As shown by their relative
resistance values, compound 15f is able to completely overcome
paclitaxel resistance (1.8 fold) as compared to 20-fold resistance
exhibited by PTX. Compound 16f is able to overcome paclitaxel
resistance by 50%, while both compounds are 50 to 90 times
more active than PTX against the epothilone-resistant 1A9-A8
cells. The relative potencies of these two compounds are unique.
The relative resistance values (RR) for 1A9-PTX10 cells
across the compounds listed in Table 2 range from 2 to 21;
those for 1A9-A8, from 3 to 82. The same quantities for 15f
and 16f are perfectly normal by comparison, similar to PTX,
Resistance and the Bridged Analogues. An important
discovery of the present work is that some of the bridged PTX
analogues display promising activity against paclitaxel-resistant
and epothilone A-resistant cell lines. The latter were derived
by long-term exposure of 1A9 human ovarian carcinoma cells
to PTX or epothilone-A (epo-A) in order to generate cells with
drug-resistant clones. Subsequent molecular characterization of

720 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Scheme 8. Synthesis of Bridged Paclitaxels 31 and 32a
Ganesh et al.
our initial conformational expectations have been fulfilled.
However, we still lack an experimental structure of a bridged-
taxane/tubulin complex and, thus, structural verification of the
idea. We note that the two-carbon bridges between the C-3′
and C-4 positions now enforce a 17-membered ring. While the
latter involves two lactones, two CdC units, and seven bonds
rigidified by the baccatin core, it is possible that the newly
installed macrocyclic rings might sustain sufficient flexibility
to require considerable conformational reorganization upon
binding to tubulin. To examine this question, we performed
empirical conformational analyses for two of our most active
analogues, 15f and 18f. For both compounds, a quantitative
NMR-ROESY determination was carried out. The corresponding
cross-peaks were translated into intramolecular proton-proton
separations based on internal distance standards. The corre-
sponding distances were combined with taxane conformers
derived by Monte Carlo conformational analyses within the
NAMFIS framework.35 As described in numerous previous
studies, this technique is able to deconvolute an averaged NMR
spectrum into a description of the ensemble of contributing
conformations along with an estimate of the individual con-
former populations.22,57
a a. LHMDS, THF, 90%; b. Second generation Grubbs catalyst, CH2Cl2,
RT, 70%; c. HF/Py, THF, 80%; d. H2, Pd/C, MeOH, 60%.
As applied to 15f with a cis double bond within the bridge,
NAMFIS analysis, using 19 NMR-ROE-derived intramolecular
atomic distances, delivered five conformations with estimated
populations of 40, 27, 17, 9, and 7% (see Experimental Section).
The top populated conformer is the T-form (40%), while the
second and fourth most populated structures (27 and 9%,
respectively) are slightly collapsed forms of the T-conformation
in which the C3′ benzamido phenyl to C2 phenyl centroid
distance is 3 Å shorter. The third and fifth structures (17 and
7%, respectively) structures are somewhat extended structures
that can be classified as neither T nor polar forms. Compound
18f delivers a similar result, namely only three conformations
from 15 ROE cross-peaks. One is the T-conformation (53%),
the second, a slightly collapsed T-structure (33%), and the third,
the polar conformer (14%). A similar analysis for parent PTX
(1a) delivered T-conformers with much reduced populations of
2-5%.37 Short-bridge ortho-tethering for this compound class
increases the concentrations of the apparent bioactive conformers
by 5-25 fold and thereby contributes to their exceptional
activity. Figure 2 illustrates the superposition of PTX and 15f
in the â-tubulin binding cleft. The conformations of both
molecules place the C-3′ benzamido and C-2 benzoyl phenyls
on either side of His227 (no π-stacking implied) and simulta-
neously avoid steric interaction with Phe270.49
Table 2. Bioactivity of Bridged Taxoids against Paclitaxel and
Epothilone A Resistant Cell Linesa
IC50, nMb
1A9-PTX10
(Fâ270V) PTX10/1A9
RRc
IC50, nMb
RRc
bridged
taxanes
1A9-A8
(Tâ274I)
1A9
A8/1A9
PTX
13g
13h
15c
15d
15f
15g
15h
16e
16f
4.8 ( 4.5
1.8 ( 2.5
157
65
42
126
157
0.13
196
157
35.9
1.03
20
18
5.5
17
9.2
1.8
6.3
21
1.8
12
21.5 ( 11.5
148 ( 67.9
137 ( 28.3
26.5 ( 3.2
46.4 ( 13.6
0.27 ( 0.08
4.5
82
10
3.8
3.8
0.84
13.7 ( 8.6
7.0 ( 0.85
12.2 ( 7.0
0.32 ( 0.35
24.6 ( 9.0
12.9 ( 7.5
18.9 ( 1.27
0.07 ( 0.02
>300 ( 0.0 >12
>300 ( 0.0 >23
66.5 ( 5.0
3.5
6.3
0.44 ( 0.19
a 1A9 is the parental drug-sensitive cell line, 1A9-PTX10 is the paclitaxel
resistant clone with an acquired Fâ270V (ref 49) mutation, and 1A9-A8 is
the epothilone A resistant clone with an acquired Tâ274I mutation which
also confers cross-resistance to paclitaxel (5-10 times). b The IC50 values
(nM) for each compound were determined in a 72-h growth inhibition assay
using the sulforhodamine-B method as previously described (ref 55). All
values represent the average of three or four independent experiments. c RR,
relative resistance ) IC50 for resistant cell line/IC50 for parental cell line.
Conclusions and Perspectives
and found in the windows RR ) 0.8-2 and 6.8-12, respec-
tively. The implication is that the bridged compounds, from the
point of view of acquired resistance, are normal taxanes. The
remarkable improvements of 15f and 16f over paclitaxel in
apparently overcoming resistance owe their origin to an unusu-
ally high potency rather than to subtle structural features of the
bridged molecules that enable them to bypass the mutated
binding site residues.
Bridged Taxane Conformations in Solution. At the outset
of our bridging studies, we hypothesized that creation of short
bridges between the ortho-position of C-3′ and the methyl of
C-4 OAc as depicted in Figure 1 would lead to bioactive
conformations in the T-Taxol family and provide highly active
taxane analogues. Table 1 provides ample support for the
concept and reinforces the proposition that deviations from ortho
substitution and short tethers evoke a reduction in both
cytotoxicity and tubulin polymerization. It would appear that
Over the past decade numerous bridging strategies have been
explored in the effort to restrain the conformation of the
paclitaxel architecture to its bioactive tubulin-bound conforma-
tion. Most have led to the synthesis of compounds that are
considerably less potent than PTX.33 A very few have demon-
strated diminished cytotoxicity but matched the latter in a
tubulin/microtuble in Vitro assay.32 Our strategy was directed
from the start by analysis of the T-Taxol conformation proposed
as the binding geometry of PTX on â-tubulin.36 Significantly,
as depicted in Figure 1, it was predicted that a bridge between
the methyl group of the C-4 acetate and the ortho-position of
the C-3′ phenyl group would lead to compounds that are
constrained to adopt the bioactive molecular shape.
In the context of activity against A2780 ovarian cancer cells,
the synthetic effort has led to seven compounds with activities
equal to that of PTX (15c,d, 16c, 25, 26, 28, and 31a), one that

Synthesis and SAR Studies of the BioactiVe Paclitaxel Conformation
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 721
is 3-fold more active (32b), three that are 30-fold enhanced (16f,
18f, 31b), and one that shows 50-fold improvement (15f). In
the PC3 prostate cell line, the activity enhancements are
somewhat less but are still significant (Table 1). Equally
important, a subset of these compounds has been tested against
both PTX- and epothilone-resistant cell lines (1A9-PTX10 and
1A9-A8, respectively, Table 2). Two of the most active
compounds, 15f and 16f, are 1200- and 160-fold more active
than PTX with respect to PTX10, respectively, and 90- and 50-
fold more cytotoxic, respectively, against cells raised against
epothilone-resistant cells (A8). Clearly, significant increases in
antiproliferative activity can have a dramatic effect on resistance
stemming from acquired mutations.
We have examined the origin of the bioactivities by seeking
to define the degree of rigidification introduced by the C-4 to
C-3′ bridging principle. This has taken the form of extracting
individual conformations associated with approximate popula-
tions from the averaged NMR spectrum of a compound. As
applied to unconstrained PTX, the T-form was estimated to be
present to the extent of 2-5% among eight diverse conforma-
tions.23,37 Constrained compounds 15f and 18f, on the other
hand, appear in solution as five and three conformations,
respectively, with 76% and 86% contributions from T-Taxol
structures. While the significant activity increases measured for
these substances cannot be attributed completely to conforma-
tional biasing, the results strongly suggest that this is a
dominating factor.
Figure 3. T-Conformation of PTX (blue) bound to â-tubulin. The
T-conformers for the analogues E-15c (green), Z-15g (yellow), and
E-18f (magenta) have been derived from NAMFIS analyses. The
baccatin cores for PTX and the three analogues are superimposed. The
meta-Z-bridged 15g (yellow) is pushed out of the taxane binding site
because phenylalanine 270 of tubulin (orange) is in steric conflict with
the extended bridge, preventing effective binding to the protein. 15c
and 18f avoid the Phe270 steric clash and fit nicely into the PTX binding
site.
Representative Procedure for the Synthesis of â-Lactam
Derivatives 7a-f. A typical procedure is described for the synthesis
of these â-lactam derivatives.
If a given compound adopts the bioactive form, does this
guarantee amplified activity? In a recent review of bridged
taxanes we pointed out that enforcing the T-Taxol conformation
is a necessary but not sufficient condition for eliciting high levels
of drug potency.33 In addition to the appropriate molecular
conformation, the tubulin-taxane ligand must also adopt a
compatible molecular volume. Compound 15g with a 5-atom
bridge between the meta position of the C-3′ phenyl and the
C-4 methyl carbon (m-O-CH2-CHdCH-CH2) illustrates the
molecular dilemma. Like PTX it displays about 5% of the
T-form in solution.38 Unlike PTX, the compound is 4-45 fold
less potent depending on cell line and 10-fold weaker as a
tubulin polymerization agent. Modeling demonstrates that a
section of the long, suboptimally substituted bridge falls outside
the molecular volume of PTX and competes with tubulin’s
Phe27049 for the same space within the binding pocket, as
illustrated by Figure 3. Consequently, the ligand either rides
higher in the pocket or is pushed out of it.38 By contrast,
compound 15c (Table 1) with a three carbon ortho-bridge (o-
O-CH2-Cd) likewise presents the T-form in solution, but the
truncated bridge avoids a steric clash with the protein and results
in activities equivalent to that of PTX.37
(a) Synthesis of Racemic 1-(p-Methoxyphenyl)-3-acetyloxy-
4-(m-allyloxyphenyl)azetidin-2-one (3a). To a solution of 3-(al-
lyloxy)benzaldehyde (1a, 5 g, 34 mmol) in CH2Cl2 (85 mL) was
added p-anisidine (4.38 g, 35 mmol), followed by MgSO4 (50 g,
in portions), and the resulting reaction mixture was stirred for 12
h. Removal of the MgSO4 by filtration and concentration of the
CH2Cl2 solution yielded crude imine 2a (8.5 g). Diisopropylethyl-
amine (100 mmol, 3 equiv) followed by acetoxyacetyl chloride (40
mmol, 1.2 equiv) were added to a solution of 2a (8.5 g, 33 mmol)
in CH2Cl2 (75 mL) at -78 °C, and the resulting solution was
brought to room temperature over 12 h. The reaction mixture was
concentrated, and the residue was purified by chromatography on
silica gel using 10-20% EtOAc in hexane to furnish racemic 1-(p-
methoxyphenyl)-3-acetyloxy-4-(m-allyloxyphenyl)azetidin-2-one (3a,
9.3 g, 75%).
(b) Resolution of 3a. Lipase PS (Amano) (2 g) was added to a
solution of 3a (4 g, 10 mmol) in CH3CN and pH 7 phosphate buffer
(1:2.5), and the resulting solution was stirred at rt for 74 h. After
usual workup the residue was separated by chromatography on silica
gel to furnish (3R,4S)-1-methoxyphenyl-3-acetyloxy-4-(m-allyl-
oxyphenyl)azetidin-2-one (4a) (1.92 g, 48%) and (3S,4R)-1-
methoxyphenyl-3-hydroxy-4-(m-allyloxyphenyl)azetidin-2-one (1.84
g, 46%).
(c) Synthesis of (3R,4S)-1-Methoxyphenyl-3-triisopropylsilyl-
oxy-4-(m-allyloxyphenyl)-azetidin -2-one (5a). A solution of 4a
(920 mg, 2.5 mmol) in tetrahydrofuran (20 mL) was added dropwise
to a stirred solution of 1 M KOH (1 M, 80 mL) and tetrahydrofuran
(20 mL), and the resulting solution was stirred for 15 min. Addition
of H2O followed by usual workup produced (3R,4S)-1-methoxy-
phenyl-3-hydroxy-4-(m-allyloxyphenyl)azetidin-2-one (800 mg,
99%). To a solution of this alcohol (800 mg, 2.48 mmol) in N,N-
dimethylformamide (3 mL) was added imidazole (500 mg, 7.4
mmol, 3 equiv) followed by Pri3SiCl (508 mg, 2.65 mmol, 1.1
equiv), and the resulting solution was stirred at room temperature
for 8 h. EtOAc was added to quench the reaction, and the usual
workup followed by chromatography over silica gel with 2% EtOAc
in hexane furnished (3R,4S)-1-methoxyphenyl-3-triisopropylsilyl-
oxy-4-(m-allyloxyphenyl)azetidin-2-one (5a) (1.15 g, 99%).
(d) Synthesis of (3R,4S)-1-Benzoyl-3-triisopropylsilyloxy-4-
(m-allyloxyphenyl)azetidin-2-one (7a). To a solution of 5a (980
mg, 2.3 mmol) in CH3CN (35 mL) was added ceric ammonium
Experimental Section
General Experimental Methods. All reagents and solvents
received from commercial sources were used without further
purification. 1H and 13C NMR spectra were obtained in CDCl3 on
Varian Unity or Varian Inova spectrometers at 400 MHz or a JEOL
Eclipse spectrometer at 500 MHz. High-resolution FAB mass
spectra were obtained on a JEOL HX-110 instrument. Specific
rotations were measured on a Perkin-Elmer 241 polarimeter. Usual
workup was carried out by quenching the reaction, extracting the
reaction mixture three times with EtOAc, combining the organic
extracts, and washing the combined extracts with H2O and brine,
followed by drying over Na2SO4, filtration, and concentration of
the organic solution to give crude product. MacroModel v7.2 and
Maestro software were obtained from Schro¨diner LLC (Portland,
OR).

722 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Ganesh et al.
nitrate (3.35 g, 6.1 mmol, 3 equiv) in H2O (17 mL). The mixture
was allowed to stand for 20 min at 0 °C and was then stirred for
45 min. EtOAc was added, and the organic phase was washed with
saturated sodium metabisulfite. Usual workup followed by chro-
matography over silica gel with 10% EtOAc in hexane furnished
the imide (3R,4S)-3-triisopropylsilyloxy-4-(m-allyloxyphenyl)aze-
tidin-2-one (6a, 600 mg, 78%). To a solution of 6a (568 mg, 1.5
mmol) in CH2Cl2 (20 mL) were added triethylamine (0.63 mL, 4.5
mmol, 3 equiv), dimethylaminopyridine (20 mg), and benzoyl
chloride (0.52 mL, 4.5 mmol, 3 equiv) at 0 °C, and the resulting
solution was stirred for 1 h. Usual workup followed by chroma-
tography over silica gel with 2-5% EtOAc furnished (3R,4S)-1-
benzoyl-3-O-triisopropylsilyloxy-4-(m-allyloxyphenyl)azetidin-2-
to quench the reaction followed by EtOAc (40 mL). The organic
phase was worked up in the usual way to give a crude product
which was subjected to preparative thin layer chromatography over
silica gel with 45% EtOAc in hexane to give 11a as a white solid
(40 mg, 72%). (11a): 1H NMR (500 MHz, CDCl3) δ ) 8.11 (2H,
d, J ) 7.1 Hz), 7.59 (1H, t, J ) 7.2 Hz), 7.46 (2H, t, J ) 7.2 Hz),
6.52 (1H, dd, J ) 17.4, 1.2 Hz, 1H), 6.47 (1H, s), 6.28 (1H, dd, J
) 17.4, 10.5 Hz), 6.01 (1H, dd, J ) 17.4, 1.2 Hz), 5.64 (1H, d, J
) 6.8 Hz), 4.94 (1H, d, J ) 7.7 Hz), 4.76 (1H, m), 4.53 (1H, dd,
J ) 10, 6.7 Hz), 4.33 (1H, d, J ) 8.4 Hz), 4.20 (1H, d, J ) 8.4
Hz), 3.94 (1H, d, J ) 6.8 Hz), 2.55 (1H, m), 2.20 (3H, s), 2.22-
2.10 (2H, m), 2.18 (3H, s), 1.90 (1H, m), 1.70 (3H, s), 1.18 (3H,
s), 1.02 (3H, s), 0.91 (9H, t, J ) 7.2 Hz), 0.60 (6H, m). 13C NMR
(125 MHz) δ ) 202.2, 169.4, 167.1, 165.3, 144.0, 133.7, 131.2,
130.1, 129.8, 129.6, 128.6, 84.2, 81.3, 78.8, 76.6, 75.8, 74.8, 72.4,
68.1, 58.8, 47.2, 42.8, 38.9, 37.3, 26.9, 21.0, 20.1, 14.9, 10.0, 6.84,
5.36. HRFABMS m/z calcd for C38H53O11Si+ 713.3357, found
713.3326 (∆ ) 4.4 ppm). Characterization data for compounds
11b-d are provided in the Supporting Information.
1
one 7a (600 mg, 82%). 7a: [R]D +97 (c ) 0.4, CHCl3). H NMR
(400 MHz) δ ) 8.02 (2H, d, J ) 7.3 Hz), 7.58 (1H, t, J ) 7.3 Hz),
7.47 (2H, t, J ) 7.4 Hz), 7.26 (1H, m), 6.98 (1H, d, J ) 7.8 Hz),
6.94 (1H, dd, J ) 1.9, 1.8 Hz), 6.86 (1H, dd, J ) 7.3, 2.5 Hz),
6.02 (1H, m), 5.39 (1H, d, J ) 6.1 Hz), 5.38 (1H, 2 × dd, J ) 16,
6 Hz), 5.26 (1H, 2 × dd, J ) 10.3, 1.6 Hz), 5.23 (1H, d, J ) 6.1
Hz), 4.51 (2H, m), 1.00 (3H, m), 0.91 (4 s, 18H). 13C NMR (125
MHz) δ ) 166.3, 165.4, 158.6, 135.4, 133.4, 133.3, 132.1, 129.9,
129.2, 128.2, 120.9, 117.6, 114.8, 114.7, 76.7, 68.9, 61.1, 17.5,
17.4, 11.7. HRFABMS: m/z calcd for C28H37NO4Si+ 479.2492,
found 479.2494 (∆ ) 0.5 ppm). Characterization data for com-
pounds 7b-d and 7f,g are provided in the Supporting Information.
Representative Procedure for the Synthesis of Baccatin III
Derivatives 11a-d. Synthesis of 4-Deacetyl-4-acryloyl-7-O-
triethylsilylbaccatin III (11a).
Representative Procedures for the Coupling of Baccatin III
Derivatives 11a-d with â-Lactam Derivatives 7a-d and 7f-g. A
typical procedure is described for the synthesis of 3′-dephenyl-3′-
(m-allyloxyphenyl)-4-deacetyl-4-acryloyl-7-O-triethylsilyl-2′-O-tri-
isopropylsilylpaclitaxel (12a).
Synthesis of 3′-Dephenyl-3′-(m-allyloxyphenyl)-4-deacetyl-4-
acryloyl-7-O-triethylsilyl-2′-O-triisopropylsilylpaclitaxel (12a).
To a solution of baccatin III derivative 11a (25 mg, 0.035 mmol)
and â-lactam derivative 7a (34 mg, 0.07 mmol, 2 equiv) in THF
(6.5 mL) was added LiHMDS at -40 °C, and the resulting solution
was stirred for 3 h. Saturated aqueous NH4Cl (2 mL) was added to
quench the reaction, and usual workup gave crude product which
was purified by preparative TLC using 20% EtOAc in hexane as
solvent to give 12a (17 mg, 65%). 1H NMR (400 MHz) δ ) 8.10
(2H, dd, J ) 7.2, 1.6 Hz), 8.00 (1H, m), 7.73 (2H, dd, J ) 7.2, 1.6
Hz), 7.70-7.30 (8H, m), 7.04 (1H, d, J ) 8.8 Hz), 6.98 (1H, d, J
) 8 Hz), 6.90 (1H, bs), 6.85 (1H, d, J ) 8 Hz), 6.63(1H, d, J )
17.6 Hz), 6.52 (1H, d, J ) 18.4 Hz), 6.47 (1H, s), 6.44 (1H, d, J
) 10.4 Hz), 6.16 (1H, t, J ) 7 Hz), 6.03 (2H, m), 5.93 (1H, d, J
) 10.8 Hz), 5.72 (1H, d, J ) 8 Hz), 5.59 (1H, d, J ) 8.8 Hz),
5.42(1H, dt, J ) 17.2, 1 Hz), 5.29 (1H, dt, J ) 10.4, 1 Hz), 4.90
(1H, d, J ) 9.6 Hz), 4.87 (1H, s), 4.65 (2H, m), 4.33 (1H, d, J )
8.8 Hz), 4.26 (1H, d, J ) 8.8 Hz), 3.90 (1H, d, J ) 7.2 Hz), 2.58
(1H, m), 2.40 (1H, m), 2.17 (3H, s), 2.10(1H, m), 2.08 (3H, s),
1.92 (1H, m), 1.74 (3H, s), 1.62 (3H, s), 1.22 (3H, s), 1.16 (3H, s),
0.95 (30H, m), 0.59 (6H, m). 13C NMR (100 MHz) δ ) 202.0,
172.0, 169.5, 167.2, 167.0, 165.3, 159.2, 140.5, 140.4, 134.2, 133.7,
133.6, 133.3, 133.0, 131.9, 130.4, 130.1, 129.8, 129.6, 129.2, 128.9,
128.7, 127.7, 127.1, 118.9, 117.9, 114.5, 113.2, 84.4, 81.5, 79.0,
76.8, 75.3, 75.28, 75.22, 72.4, 71.7, 69.1, 58.6, 55.9, 47.0, 43.5,
37.4, 26.7, 21.8, 21.1, 18.0, 14.4, 12.7, 10.4, 6.9, 5.5. HRFABMS
m/z calcd for C66H89NO15Si2Na+ 1214.5668, found 1214.5668 (∆
) 0 ppm). Characterization data for compounds 12b-f are provided
in the Supporting Information.
(a) Synthesis of 1-Dimethylsilyl-4-deacetyl-4-acryloyl-4-
deacetyl-7-O,10-O,13-O-tris-triethylsilylbaccatin III (9a). Lithium
hexamethyldisilazide (LiHMDS) (1 M, 1.3 mL, 1.3 mmol, 1.2
equiv) was added dropwise at 0 °C to a solution of 1-dimethylsilyl-
4-deacetyl-7-O,10-O,13-O-tris-triethylsilylbaccatin III46 (8) (1 g, 1.1
mmol) in THF (6 mL), and the resulting solution was stirred for
45 min. Acryloyl chloride (0.123 mL, 1.54 mmol, 1.3 equiv) was
added to the above solution at 0 °C, and the reaction mixture stirred
for 3 h. Saturated NH4Cl solution (10 mL) was added, and the
aqueous phase was subjected to the usual workup. The crude
product as purified by chromatography over silica gel with 4%
EtOAc in hexane to furnish 1-dimethylsilyl-4-deacetyl-4-acryloyl-
7-O,10-O,13-O-tris-triethylsilylbaccatin III (9a) as a white solid
(550 mg, 52%).
(b) Synthesis of 4-Deacetyl-4-acryloyl-10-deacetylbaccatin III.
To a solution of 1-dimethylsilyl-4-deacetyl-4-acryloyl-7-O,10-O,13-
O-tris-triethylsilylbaccatin III (9a) (470 mg, 0.49 mmol) in THF
(30 mL) was added dropwise HF·Py (70% HF, 2.37 mL) at 0 °C,
and the resulting solution was brought to room temperature over
24 h, after which saturated NaHCO3 solution (50 mL) was added
carefully to quench the reaction. The aqueous phase was subjected
to the usual workup, and the crude product was purified by
preparative thin layer chromatography on silica gel developed with
70% EtOAc in hexane to give 4-deacetyl-4-acryloyl-10-deacetyl-
baccatin III as a white solid (193 mg, 70%).
(c) Synthesis of 4-Deacetyl-4-acryloylbaccatin III (10a).
Anhydrous CeCl3 (20 mg) was added to a solution of 4-deacetyl-
4-acryloyl-10-deacetyl-baccatin III (210 mg, 0.377 mmol) in THF
(10 mL) and the resulting solution was stirred for 5 min. Acetic
anhydride (0.65 mL, 14-fold excess) was added to the above
solution and the reaction mixture was stirred for 4 h. EtOAc (100
mL) was added and the solution was washed with saturated
NaHCO3, water, and brine, dried over Na2SO4, and concentrated.
The crude product was subjected to preparative thin layer chro-
matography on silica gel with 60% EtOAc in hexane to give
4-deacetyl-4-acryloyl-baccatin III (10a) as a white solid (200 mg,
96%).
(d) Synthesis of 4-Deacetyl-4-acryloyl-7-O-triethylsilylbacca-
tin III (11a). To a solution of 4-deacetyl-4-acryloylbaccatin III
(10a) (50 mg, 0.083 mmol) in dichloromethane (5 mL) was added
imidazole (56 mg, 0.836 mmol, 10 equiv) followed by triethylsilyl
chloride (0.50 mmol, 6 equiv) at 0 °C, and the resulting solution
was stirred for 3 h. Dilute HCl (0.05M, 5 mL) solution was added
Representative Procedure for the Deprotection of 12a-i.
Synthesis of 3′-Dephenyl-3′-(m-allyloxyphenyl)-4-deacetyl-4-
acryloylpaclitaxel (13a). To a solution of 3′-dephenyl-3′-(m-
allyloxyphenyl)-4-deacetyl-4-acryloyl-7-O-triethylsilyl-2′-O-triiso-
propylsilylpaclitaxel (12a, 15 mg, 0.012 mmol) in THF (5 mL)
was added hydrogen fluoride-pyridine complex (70%, 0.2 mL) at
0 °C, and the resulting solution was brought to room temperature
overnight. Saturated NaHCO3 solution (5 mL) was added carefully
to quench the reaction. Usual workup followed by preparative TLC
(silica gel, 50% EtOAc in hexane) furnished 13a (5.5 mg, 55%).
(13a): 1H NMR (400 MHz, CDCl3) δ ) 8.15 (2H, d, J ) 7.2 Hz),
7.72 (2H, d, J ) 8 Hz), 7.61 (1H, t, J ) 7 Hz), 7.50 (3H, m), 7.40
(3H, m), 7.10 (2H, m), 6.91(1H, d, J ) 7.6 Hz,) 6.83 (1H, d, J )
9.2 Hz), 6.50 (1H, dd, J ) 17.6, 1.2 Hz), 6.35 (1H, d, J ) 10.4
Hz), 6.29 (1H, s), 6.1 (1H, m), 6.05 (1H, m), 5.68 (3H, m), 5.47
(1H, dd, J ) 9.2, 1.6 Hz), 5.30 (1H, dq, J ) 17.2, 1.6 Hz), 5.30
(1H, dq, J ) 10.5, 1.2 Hz), 4.92 (1H, dd, J ) 9.4, 1.6 Hz), 4.74
(1H, d, J ) 2 Hz), 4.56 (2H, m), 4.48 (1H, dd, J ) 10.4, 6.8 Hz),

Synthesis and SAR Studies of the BioactiVe Paclitaxel Conformation
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 723
4.34 (1H, d, J ) 8.4 Hz), 4.25 (1H, d, J ) 8.4 Hz), 3.86 (1H, d,
J ) 6.8 Hz), 2.58 (1H, m), 2.42 (1H, m), 2.30 (1H, m), 2.24 (3H,
s), 1.90 (1H, m), 1.83 (3H, s), 1.70 (3H, s), 1.62 (1H, m), 1.22
(3H, s), 1.14 (3H, s). 13C NMR (100 MHz) δ ) 203.8, 173.3, 171.5,
167.2, 167.1, 165.4, 159.3, 142.3, 140.1, 133.9, 133.4, 133.2, 132.4,
132.1, 130.4, 130.2, 129.5, 129.3, 128.94, 128.91, 127.2, 119.6,
118.2, 114.5, 114.0, 84.6, 81.6, 79.1, 76.7, 75.8, 75.2, 72.7, 72.3,
72.2, 69.1, 58.8, 54.5, 45.9, 43.3, 35.9, 35.7, 32.1, 29.9, 27.0, 24.9,
concentrated to crude mass, which was applied to silica gel
preparative plate using 65% EtOAc in hexane as solvent provided
the dihydro bridged paclitaxel 16a (5 mg, 83% yield). 16a: 1H NMR
(400 MHz) δ ) 8.06 (2H, d, J ) 7.2 Hz), 7.82 (2H, d, J ) 7.2
Hz), 7.10-7.03 (4H, m), 7.55-7.45 (4H, m), 7.19 (1H, t, J ) 7.6
Hz), 7.08 (1H, bs), 6.96-6.88 (2H, m), 6.19 (1H, s), 6.14 (1H, t,
J ) 8.8 Hz) 5.57 (1H, d, J ) 7.2 Hz), 5.24 (1H, dd, J ) 10.6, 6.8
Hz), 4.89 (1H, d, J ) 8.4 Hz), 4.62 (1H, m), 4.42 (2H, m), 4.32
(1H, m), 4.22 (1H, d, J ) 7.2 Hz), 3.52 (1H, d, J ) 6.8 Hz), 2.70
(1H, m), 2.50 (5H, m), 2.23 (1H, m), 2.22 (3H, s), 2.04 (3H, s),
1.88 (1H, m), 1.65 (3H, s), 1.60 (4H, m), 1.42 (3H, s), 1.10 (3H,
s). 13C NMR (100 MHz) δ ) 203.6, 172.9, 171.5, 170.7, 167.0,
159.7, 142.7, 137.6, 134.2, 133.6, 133.5, 132.8, 132.4, 131.3, 130.4,
129.4, 129.07, 129.0, 127.4, 127.3, 122.1, 119.0, 109.6, 84.4, 80.9,
79.6, 76.3, 75.6, 74.9, 72.4, 70.5, 64.9, 64.5, 60.6, 59.7, 58.4, 45.7,
43.2, 38.3, 35.5, 35.1, 32.1, 31.4, 30.8, 29.92, 29.89, 29.5, 27.8,
27.1, 22.5, 21.0, 19.3, 14.7, 14.3, 13.9, 9.7. HRFABMS m/z calcd
for C49H54NO15Na+ 918.3313, found 918.3337 (∆ ) 2.6 ppm).
NAMFIS Analysis: 2D NMR-ROESY Spectra for 15f and
18f. The 1D 1H assignments of 15f and 18f were accomplished by
interpretation of the corresponding COSY, HMBC, and HSQC
spectra. The 2D NMR ROESY analysis for both compounds was
performed on an INOVA 400 MHz NMR spectrometer with 70,
100, 125, 150, 180 ms mixing times to check linearity of the cross-
relaxation buildup rates. Interproton distances were calculated from
the integrated cross-peak volumes using the initial rate approxima-
tion and an internal calibration distance between H-6a and H-6b
of 1.77 Å. This provided 19 and 15 ROE-based interproton distances
for 15f and 18f, respectively.
Solution Conformation Deconvolution for 15f and 18f. A
10 000-step conformational search was performed on both mol-
ecules with the MMFF94s force field and the GBSA/H2O solvation
model in MacroModel v7.2. An energy cutoff of 12 kcal/mol
resulted in a pool of 93 and 374 unique conformations for 15f and
18f, respectively, with the global minimum found 62 and 29 times,
respectively. The NAMFIS methodology integrated 19 ROE-
determined distances measured in CDCl3 and the 93 conformers
of 15f to deconvolute the NMR data into four conformations. One
of these, however, places the C-4 acetate CdO beneath the oxetane
ring. Inspection of literature X-ray structures of PTX and analogues
shows that the carbonyl group consistently resides beneath the six-
membered C-ring. Quantum chemical calculations (B3LYP/6-31G*,
not shown) also demonstrate that oxetane orientation is ca. 4 kcal/
mol higher in energy. Thus the 93 conformers were depleted of
the latter conformation, and NAMFIS was rerun to give five
conformations with populations of 40, 27, 17, 9, and 7%. (SSD )
59). The dominant conformer is the T-form (40%), while the second
and fourth most populated structures (27 and 9%, respectively) are
slightly collapsed variants of the T-conformation in which the C3′-
benzamido phenyl to C2-phenyl centroid distance is 3 Å shorter.
The third and fifth conformers (17 and 7%, respectively) are
somewhat extended structures that can be classified as neither T
nor polar forms. For 18f, the 374 conformations likewise included
a subset of carbonyl/oxetane structures, one of which appeared in
the initial NAMFIS run employing 15 ROE-determined distances.
These high-energy structures were removed from the dataset. A
second NAMFIS run yielded three conformations with populations
of 53, 33, and 14% (SSD ) 96). One is the T-conformation (53%),
the second, a slightly collapsed T-structure (33%), and the third,
the polar conformer (14%).
+
22.9, 22.0, 21.0, 15.1, 9.8. HRFABMS m/z calcd for C51H56NO15
922.3650, found 922.36676 (∆ ) 2.9 ppm). Similar procedures
were employed to prepare compounds 13b-j; characterization data
for these compounds are provided in the Supporting Information.
Representative Procedure for Ring-Closing Metathesis of
12a-i. Synthesis of 7-O-Triethylsilyl-2′-O-triisopropylsilyl Bridged
Paclitaxel 14a. Grubbs second generation catalyst (2 mg) in CH2-
Cl2 (2.5 mL) was added over 3 h to a solution of 12a (16 mg,
0.013 mmol) in CH2Cl2 (4 mL), and the resulting solution was
stirred for 1 h. The reaction mixture was concentrated, and the crude
mass was applied to preparative thin layer chromatography using
25% EtOAc in hexane as solvent to furnish 14a (10 mg, 65%).
14a: 1H NMR (500 MHz) δ ) 8.10 (2H, d, J ) 7.1 Hz), 7.82 (2H,
d, J ) 7.1 Hz), 7.65 (1H, t, J ) 7.3 Hz), 7.53 (2H, m), 7.45 (3H,
m), 7.35 (1H, m), 7.21 (2H, m), 6.94 (2H, m), 6.46 (1H, s), 6.14
(1H, m), 6.12 (1H, d, J ) 16 Hz), 5.73 (1H, d, J ) 7.3 Hz), 5.5
(1H, d, J ) 6.6 Hz), 4.93 (2H, m), 4.85 (1H, d, J ) 8 Hz), 4.56
(1H, d, J ) 2 Hz), 4.48 (1H, dd, J ) 10.5, 6.6 Hz), 4.28 (2H, s),
3.81 (1H, d, J ) 7.1 Hz), 2.50 (1H, m), 2.29 (1H, m), 2.18 (3H, s),
2.16 (1H, m), 2.08 (3H, s), 1.99 (3H, m), 1.91 (1H, m), 1.74 (3H,
s), 1.24 (3H, s), 1.17 (3H, s), 1.10 (21H, m), 0.9 (9H, t, J ) 7.8
Hz), 0.57 (6H, m). 13C NMR (125 MHz) δ ) 201.8, 171.5, 169.5,
167.3, 167.1, 164.1, 159.7, 147.0, 142.6, 140.5, 134.1, 134.0, 133.6,
132.0, 130.1, 130.0, 129.7, 128.9, 127.2, 123.8, 118.9, 118.2, 113.7,
84.4, 81.9, 79.1, 77.0, 75.2, 75.1, 72.7, 70.8, 70.0, 58.7, 57.6, 47.6,
43.4, 37.4, 35.9, 26.8, 21.6, 21.1, 18.1, 17.9, 14.5, 12.9, 10.3, 6.9,
5.5. HRFABMS m/z calcd for C64H85NO15Si2Na+ 1186.5355, found
1185.5330 (∆ ) 2.2 ppm). Similar procedures were employed to
prepare compounds 14b-i; characterization data for these com-
pounds are provided in the Supporting Information.
Representative Procedure for the Deprotection of Macrocy-
clic Paclitaxels 14a-j. Synthesis of Bridged Paclitaxel 15a. To
a solution of 14a (9 mg, 0.0077 mmol) in THF (3.5 mL) was added
hydrogen fluoride-pyridine complex (70%, 0.15 mL) at 0 °C, and
the resulting solution was brought to room temperature overnight.
Saturated NaHCO3 solution (5 mL) was added carefully to quench
the reaction and after the usual workup resulted the crude product
was purified by preparative TLC (silica gel, 60% EtOAc in hexane)
to furnish 15a (6.3 mg, 91%). 15a: 1H NMR (400 MHz) δ ) 7.98
(2H, d, J ) 7.6 Hz), 7.80 (2H, d, J ) 7.6 Hz), 7.69 (1H, t, J ) 7.6
Hz), 7.61 (1H, t, J ) 6.8 Hz), 7.53 (1H, m), 7.44 (2H, m), 7.22
(1H, dd, J ) 7.6 Hz), 7.10 (1H, m), 7.05 (1H, bs), 6.98 (1H, m),
6.80 (1H, bs), 6.26 (s, 1H), 6.13 (1H, d, J ) 16 Hz) 6.11 (1H, d,
J ) 9.2 Hz), 5.60 (1H, d, J ) 6.8 Hz), 5.31 (1H, dd, J ) 9.3, 6.4
Hz), 5.04 (1H, dd, J ) 14.4, 9.6 Hz), 4.88 (2H, m), 4.48 (1H, d, J
) 9.6 Hz), 4.37 (1H, m), 4.21 (2H, s), 3.68 (1H, d, J ) 6.8 Hz),
2.58 (2H, m), 2.22 (3H, s), 1.90 (1H, m), 1.84 (3H, s), 1.78 (1H,
m), 1.68 (3H, s), 1.53 (1H, m), 1.25-1.22 (1H, m), 1.21 (3H, s),
1.0 (3H, s). 13C NMR (100 MHz) δ ) 203.7, 173.0, 171.5, 169.3,
167.0, 163.6, 155.7, 143.3, 142.9, 138.4, 134.1, 133.6, 132.6, 132.4,
131.2, 130.1, 129.6, 128.9, 127.3, 126.8, 122.5, 119.3, 111.2, 84.3,
81.5, 79.4, 77.4, 76.7, 76.4, 75.7, 75.1, 72.5, 70.5, 66.1, 58.8, 58.6,
45.9, 43.2, 35.7, 35.0, 27.0, 22.3, 21.0, 14.8, 9.7. HRFABMS m/z
calcd for C49H52NO15 894.3337, found 894.3294 (∆ ) 4.8 ppm).
Similar procedures were employed to prepare compounds 15b-j;
characterization data for these compounds are provided in the
Supporting Information.
Glide Docking. The top populated NAMFIS-derived conforma-
tion of 15f was docked into the 1JFF tubulin pocket using Glide58
in Maestro. An initial rigid dock of the ligand did not allow it to
be situated in the pocket, so a follow-up flexible dock using the
extra precision (XP) option was performed. It resulted in three very
similar poses, all in the T-shape form, with the top pose differing
from the NAMFIS conformation with an rms deviation of only 0.6
Å when all heavy atoms (C, N, O) were superposed.
Representative Procedure for the Hydrogenation of Macro-
cyclic Paclitaxels 15a-j. Synthesis of Dihydro Bridged Paclitaxel
16a. To a solution of 15a (6 mg, 0.0067 mmol) in methanol (5
mL) was added 10%Pd-C (10 mg) and hydrogenated at 35 psi for
6 h. The reaction mixture was filtered a through short plug of silica
gel, eluting with 20% methanol in EtOAc. The filtrate was
Biological Data. IC50 values were determined using the published
method59 for A2780 cells, the MTT assay60 for PC3 cells, and the
sulforhodamine-B method for the paclitaxel-resistant cell lines.55

724 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Ganesh et al.
(15) Williams, H. J.; Scott, A. I.; Dieden, R. A.; Swindell, C. S.; Chirlian,
L. E.; Francl, M. M.; Heerding, J. M.; Krauss, N. E. NMR and
Molecular Modeling Study of Active and Inactive Taxol Analogues
in Aqueous and Non Aqueous Solution. Can. J. Chem. 1994, 72,
252-60.
(16) Cachau, R. E.; Gussio, R.; Beutler, J. A.; Chmurny, G. N.; Hilton,
B. D.; Muschik, G. M.; Erickson, J. W. Solution Structure of Taxol
Determined Using a Novel Feedback-scaling Procedure for NOE-
restrained Molecular Dynamics. Supercomput. Appl. High Perform.
Comput. 1994, 8, 24-34.
(17) Vander Velde, D. G.; Georg, G. I.; Grunewald, G. L.; Gunn, C. W.;
Mitscher, L. A. “Hydrophobic Collapse” of Taxol and Taxotere
Solution Conformations in Mixtures of Water and Organic Solvent.
J. Am. Chem. Soc. 1993, 115, 11650-1.
(18) Paloma, L. G.; Guy, R. K.; Wrasidlo, W.; Nicolaou, K. C.
Conformation of a Water-soluble Derivative of Taxol in Water by
2D-NMR Spectroscopy. Chem. Biol. 1994, 1, 107-12.
ED50 values for induction of tubulin assembly were determined
using either light scattering at 350 nm in a Hewlett-Packard 8453
absorption spectrometer or fluorescence in a 96-well plate format.61
Acknowledgment. This work was supported by the National
Cancer Institute, National Institutes of Health (Grant CA-69571),
and we are grateful for this support. We are likewise grateful
to William Bebout and Rebecca Guza in the Department of
Chemistry, Virginia Polytechnic Institute and State University,
for mass spectra and preliminary cytotoxicity determinations,
respectively. We are pleased to acknowledge the support and
encouragement of Prof. Dennis Liotta (Department of Chem-
istry, Emory University).
(19) Ojima, I.; Chakravarty, S.; Inoue, T.; Lin, S.; He, L.; Horwitz, S.
B.; Kuduk, S. C.; Danishefsky, S. J. A Common Pharmacophore for
Cytotoxic Natural Products that Stabilize Microtubules. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 4256-61.
(20) Ojima, I.; Kuduk, S. D.; Chakravarty, S.; Ourevitch, M.; Begue, J-P.
A Novel Approach to the Study of Solution Structures and Dynamic
Behavior of Paclitaxel and Docetaxel Using Fluorine-Containing
Analogues as Probes. J. Am. Chem. Soc. 1997, 119, 5519-27.
(21) Ojima, I.; Inoue, T.; Chakravarty, S. Enantiopure Fluorine-containing
Taxoids: Potent Anticancer Agents and Versatile Probes for Bio-
medical Problems. J. Fluorine Chem. 1999, 97, 3-10.
(22) Snyder, J. P.; Nevins, N.; Cicero, D. O.; Jansen, J. The Conformations
of Taxol in Chloroform. J. Am. Chem. Soc. 2000, 122, 724-5.
(23) Snyder, J. P.; Nevins, N.; Jimenez-Barbero, Cicero, D.; Jansen, J.
M. Unpublished.
(24) Li, Y.; Poliks, B.; Cegelski, L.; Poliks, M.; Gryczynski, Z.; Piszczek,
G.; Jagtap, P. G.; Studelska, D. R.; Kingston, D. G. I.; Schaefer, J.;
Bane, S. Conformation of Microtubule-Bound Paclitaxel Determined
by Fluorescence Spectroscopy and REDOR NMR. Biochemistry
2000, 39, 281-291.
(25) Barboni, L.; Lambertucci, C.; Appendino, G.; Vander Velde, D. G.;
Himes, R. H.; Bombardelli, E.; Wang, M.; Snyder, J. P. Synthesis
and NMR-Driven Conformational Analysis of Taxol Analogoues
Conformationally Constrained on the C13 Side Chain. J. Med. Chem.
2001, 44, 1576-1587.
(26) Boge, T. C.; Wu, Z-J.; Himes, R. H.; Vander Velde, D. G.; Georg,
G. I. Conformationally Restricted Paclitaxel Analogues: Macrocyclic
Mimics of the “Hydrophobic Collapse” Conformation. Bioorg. Med.
Chem. Lett. 1999, 9, 3047-52.
(27) Ojima, I.; Lin, S.; Inoue, T.; Miller, M. L.; Borella, C. P.; Geng, X.;
Walsh, J. J. Macrocycle Formation by Ring-closing Metathesis.
Application to the Syntheses of Novel Macrocyclic Taxoids. J. Am.
Chem. Soc. 2000, 122, 5343-53.
Supporting Information Available: Characterization data for
new compounds 7b-d,f,g, 11b-d, 12b-k, 13b-j, 14c,d,f-k,
15c,d,f-k, 16c,d,f-j, 25, 26, 28, 31a,b, and 32a,b; 1H NMR
spectra of compounds 15c,d,f, 15f (expanded), 15f (COSY), 16c,f,
25, 31a,b, and 32a,b. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) (a) Rowinsky, E. K.; The Development and Clinical Utility of the
Taxane Class of Antimicrotuble Chemotherapy Agents. Annu. ReV.
Med. 1997, 48, 353-374. (b) Crown, J.; O’Leary, M. The Taxanes:
an Update. The Lancet 2000, 355, 1176-1178.
(2) Halkin, A.; Stone, G. W. Polymer-based Paclitaxel-eluting Stents in
Percutaneous Coronary Intervention: A Review of the TAXUS Trials.
J. InterVent. Cardiol. 2004, 17, 271-282.
(3) (a) Bollag, D. M.; McQueney, P. A.; Zhu, J.; Hensens, O.; Koupal,
L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C. M. Epothilones,
a New Class of Microtubule-stabilizing Agents with a Taxol-like
Mechanism of Action. Cancer Res. 1995, 55, 2325-2333. (b)
Kowalski, R. J.; Giannakakou, P.; Hamel, E. Activities of the
Microtubule-stabilizing Agents Epothilones A and B with Purified
Tubulin and in Cells Resistant to Paclitaxel (Taxol). J. Biol. Chem
1997, 272, 2534-2541.
(4) ter Haar, E.; Kowalski, R. J.; Hamel, E.; Lin, C. M.; Longley, R. E.;
Gunasekera, S. P.; Rosenkranz, H. S.; Day, B. W. Discodermolide,
a Cytotoxic Marine Agent that Stabilizes Microtubules More Potently
than Taxol. Biochemistry 1996, 35, 243-250.
(5) Gapud, E. J.; Bai, R.; Ghosh, A. K.; Hamel, E., Laulimalide and
Paclitaxel: A Comparison of their Effects on Tubulin Assembly and
their Synergistic Action when Present Simultaneously. Mol. Phar-
macol. 2004, 66, 113-121.
(28) Ojima, I.; Geng, X.; Lin, S.; Pera, P.; Bernacki, R. J. Design,
Synthesis and Biological Activity of Novel C2-C3.′ N-Linked
Macrocyclic Taxoids. Bioorg. Med. Chem. Lett. 2002, 12, 349-52.
(29) Geng, X.; Miller, M. L.; Lin, S.; Ojima, I. Synthesis of Novel C2-
C3.′N-Linked Macrocyclic Taxoids by Means of Highly Regio-
selective Heck Macrocyclization. Org. Lett. 2003, 5, 3733-6.
(30) Querolle, O.; Dubois, J.; Thoret, S.; Dupont, C.; Gue´ritte, F.; Gue´nard,
D. Synthesis of Novel 2-O,3′-N-Linked Macrocyclic Taxoids with
Variable Ring Size. Eur. J. Org. Chem. 2003, 542-50.
(31) Querolle, O.; Dubois, J.; Thoret, S.; Roussi, F.; Montiel-Smith, S.;
Gue´ritte, F.; Gue´nard, D. Synthesis of Novel Macrocyclic Docetaxel
Analogues. Influence of Their Macrocyclic Ring Size on Tubulin
Activity. J. Med. Chem. 2003, 46, 3623-30.
(32) (a) Querolle, O.; Dubois, J.; Thoret, S.; Roussi, F.; Gue´ritte, F.;
Gue´nard, D. Synthesis of C2-C3.′ N-Linked Macrocyclic Taxoids.
Novel Docetaxel Analogues with High Tubulin Activity. J. Med.
Chem. 2004, 47, 5937-5944. (b) Geney, R.; Sun, L.; Pera, P.;
Bernacki, R. J.; Xia, S.; Horwitz, S. B.; Simmerling, C. L.; Ojima,
I. Use of the Tubulin Bound Paclitaxel Conformation for Structure-
Based Rational Drug Design. Chem. Biol. 2005, 12, 339-348.
(33) Kingston, D. G. I.; Bane, S.; Snyder, J. P. The Taxol Pharmacophore
and the T-Taxol Bridging Principle. Cell Cycle 2005, 4, 279-289.
(34) Gue´ritte-Voegelein, F.; Gue´nard, D.; Mangatal, L.; Potier, P.;
Guilhem, J.; Ce´sario, M.; Pascard, C. Structure of a Synthetic Taxol
Precursor: N-tert-Butoxycarbonyl-10-deacetyl-N-debenzoyltaxol. Acta
Crystallogr. C 1990, 46, 781-784.
(6) Lindel, T.; Jensen, P. R.; Fenical, W.; Long, B. H.; Casazza, A. M.;
Carboni, J.; Fairchild, C. R. Eleutherobin, a New Cytotoxin that
Mimics Paclitaxel (Taxol) by Stabilizing Microtubules. J. Am. Chem.
Soc. 1997, 119, 8744-8745.
(7) Schiff, P. B.; Fant, J.; Horwitz, S. B. Promotion of Microtubule
Assembly in vitro by Taxol. Nature 1979, 277, 665-667.
(8) Horwitz, S. B. Mechanism of Action of Taxol. Trends Pharmacol.
Sci. 1992, 13, 134-136.
(9) Jordan, M. A.; Toso, R. J.; Thrower, D.; Wilson, L. Mechanism of
Mitotic Block and Inhibition of Cell Proliferation by Taxol at Low
Concentrations. Proc. Natl. Acad. Sci. 1993, 90, 9552-9556.
(10) Blagosklonny, M. V.; Fojo, T. Molecular Effects of Paclitaxel: Myths
and Reality (a Critical Review). Int. J. Cancer 1999, 83, 151-156.
(11) (a) Sunters, A.; Madureira, P. A.; Pomeranz, K. M.; Aubert, M.;
Brosens, J. J.; Cook, S. J.; Burgering, B. M.; Coombes, R. C.; Lam,
E. W. Paclitaxel-induced Nuclear Translocation of FOXO3a in Breast
Cancer Cells is Mediated by c-Jun NH2-terminal Kinase and Akt.
Cancer Res. 2006, 66, 212-220. (b) Faried, L. S.; Faried, A.;
Kanuma, T.; Nakazato, T.; Tamura, T.; Kuwano, H.; Minegishi, T.
Inhibition of the Mammalian Target of Rapamycin (mTOR) by
Rapamycin Increases Chemosensitivity of CaSki Cells to Paclitaxel.
Eur. J. Cancer 2006, 42, 934-947.
(12) Nogales, E.; Wolf, S. G.; Downing, K. H. Structure of the Râ Tubulin
Dimer by Electron Crystallography. Nature 1998, 391, 199-203.
(13) Lowe, J.; Li, H.; Downing, K. H.; Nogales, E. Refined Structure of
Râ-Tubulin at 3.5 Å Resolution. J. Mol. Biol. 2001, 313, 1045-
1057.
(14) Dubois, J.; Guenard, D.; Gueritte-Voeglein, F.; Guedira, N.; Potier,
P.; Gillet, B.; Betoeil, J-C. Conformation of Taxotere and Analogues
Determined by NMR Spectroscopy and Molecular Modeling Studies.
Tetrahedron 1993, 49, 6533-44.
(35) Cicero, D. O.; Barbato, G.; Bazzo, R.; NMR Analysis of Molecular
Flexibility in Solution: A New Method for the Study of Complex
Distributions of Rapidly Exchanging Conformations. Application to
a 13-Residue Peptide with an 8-Residue Loop. J. Am. Chem. Soc.
1995, 117, 1027-33.

Synthesis and SAR Studies of the BioactiVe Paclitaxel Conformation
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 725
(36) Snyder, J. P.; Nettles, J. H.; Cornett, B.; Downing, K. H.; Nogales,
E. The Binding Conformation of Taxol in â-Tubulin: A Model Based
on the Electron Crystallographic Density. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 5312-5316.
(37) Ganesh, T.; Guza, R. C.; Bane, S.; Ravindra, R.; Shanker, N.;
Lakdawala, A. S; Snyder, J. P.; Kingston, D. G. I. The Bioactive
Taxol Conformation on â-tubulin: Experimental Evidence from
Highly Active Constrained Analogs. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 10006-10011.
(38) Metaferia, B. B.; Hoch, J.; Glass, T. E.; Bane, S. L.; Chatterjee, S.
K.; Snyder, J. P.; Lakdawala, A.; Cornett, B.; Kingston, D. G. I.
Synthesis and Biological Evaluation of Novel Macrocyclic Paclitaxel
Analogues. Org. Lett. 2001, 3, 2461-2464.
(39) Paik, Y.; Yang, C.; Metaferia, B.; Tang, S.; Bane, S.; Ravindra, R.;
Shanker, N.;Alcaraz, A. A. Snyder, J. P.;Schaefer, J.; O’Connor, R.
D.; Cegelski, L.; Kingston, D. G. I. REDOR NMR Distance
Measurements for the Tubulin-Bound Paclitaxel Conformation. J.
Am. Chem. Soc. 2007, 129, 361-370.
(40) Tang, S.; Yang, C.; Brodie, P.; Bane, S.; Ravindra, R.; Sharma, S.;
Jiang, Y.; Snyder, J. P.; Kingston, D. G. I. Bridging Converts a
Noncytotoxic nor-Paclitaxel Derivative to a Cytotoxic Analogue by
Constraining it to the T-Taxol Conformation. Org. Lett. 2006, 8,
3983-3986.
(41) For the leading references, see: (a) Trnka, T. M.; Grubbs, R.H. The
Development of L2X2Ru)CHR Olefin Metathesis Catalysts: An
Organometallic Success Story. Acc. Chem. Res. 2001, 34, 18-29,
and references cited therein. (b) Fu¨rstner, A. Olefin Metathesis and
Beyond. Angew. Chem., Int. Ed. 2000, 39, 3012-3043. (c) Grubbs,
R. H.; Chang, S. Recent Advances in Olefin Metathesis and its
Applications in Organic Synthesis. Tetrahedron 1998, 54, 4413-
4450. and references cited therein.
(42) For the synthesis of various substituted racemic â-lactams, see: Carr,
J. A.; Al-Azemi, T. F.; Long, T. E.; Shim, J-Y.; Coates, C. M.; Turos,
E.; Bisht, K. S. Lipase Catalyzed Resolution of 4-Aryl Substituted
â-Lactams: Effect of Substitution on the 4-Aryl Ring. Tetrahedron
2003, 59, 9147-9160, and references cited therein.
(43) Brieva, R.; Crich, J. Z.; Sih, C. J. Chemoenzymatic Synthesis of the
C-13 Side Chain of Taxol: Optically Active 3-Hydroxy-4-phenyl
â-lactam Derivatives. J. Org. Chem. 1993, 58, 1068-1075.
(44) Substrates 4a-d took 3-7 days for resolution using lipase PS, and
substrate 4e took 35 days for complete resolution.
Model for the Microtubule Binding of Paclitaxel and Epothilone.
Org. Lett. 1999, 1, 43-46.
(51) Chaudhary, A. G.; Gharpure, M. M.; Rimoldi, J. M.; Chordia, M.
D.; Gunatilaka, A. A. L.; Kingston, D. G. I.; Grover, S.; Lin, C. M.;
Hamel, E. Unexpectedly Facile Hydrolysis of the 2-Benzoate Group
of Taxol and Synthesis of Analogues With Improved Activities. J.
Am. Chem. Soc. 1994, 116, 4097-4098.
(52) (a) Ojima, I.; Kuduk, S. D.; Pera, P.; Veith, J. M.; Bernacki, R. J.,
Syntheses, and Structure-Activity, Relationships, of Nonaromatic,
Taxoids: Effects, of Alkyl, and Alkenyl, Ester, Groups, on Cyto-
toxicity. J. Med. Chem. 1997, 40, 279-285. (b) Georg, G. I.;
Harriman, G. C. B.; Ali, S. M.; Datta, A.; Hepperle, M.; Himes, R.
H., Synthesis of 2-O-Heteroaroyl Taxanes: Evaluation of Microtubule
Assembly Promotion and Cytotoxicity. Bioorg. Med. Chem. Lett.
1995, 5 (2), 115-118. (c) Nicolaou, K. C.; Renaud, J.; Nantermet,
P. G.; Couladouros, E. A.; Guy, R. K.; Wrasidlo, W. Chemical
Synthesis and Biological Evaluation of C-2 Taxoids. J. Am. Chem.
Soc. 1995, 117, 2409-2420.
(53) Alcaraz, A. A.; Mehta, A. K.; Snyder, J. P. The T-Taxol Conforma-
tion. J. Med. Chem. 2006, 49, 2478-2488.
(54) Giannakakou, P.; Gussio, R.; Nogales, E.; Downing, K. H.; Zaha-
revitz, D.; Bollbuck, B.; Poy, G.; Sackett, D.; Nicolaou, K. C.; Fojo,
T. A common pharmacophore for epothilone and taxanes: Molecular
basis for drug resistance conferred by tubulin mutations in human
cancer cells. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2904-2909.
(55) Giannakakou, P.; Sackett, D.; Kang, Y.-K.; Zhan, Z.; Buters, J. T.
M.; Fojo, T.; Poruchynsky, M. S. Paclitaxel-resistant Human Ovarian
Cancer Cells have Mutant â-Tubulins That Exhibit Impaired Pali-
taxel-driven Polymerization. J. Biol. Chem. 1997, 272, 17118-17125.
(56) Zhou, J.; O’Brate, A.; Zelnak, A.; Giannakakou, P. Survivin
Deregulation in â-Tubulin Mutant Ovarian Cancer Cells Underlies
Their Compromised Mitotic Response to Taxol. Cancer Res. 2004,
64, 8708-8714.
(57) (a) Nevins, N.; Cicero, D.; Snyder, J. P. A Test of the Single-
Conformation Hypothesis in the Analysis of NMR Data for Small
Polar Molecules: A Force Field Comparison. J. Org. Chem. 1999,
64, 3979-3986. (b) Monteagudo, E.; Cicero, D. O.; Cornett, B.;
Myles, D. C.; Snyder, J. P. The Conformations of Discodermolide
in DMSO. J. Am. Chem. Soc. 2001, 123, 6929-6930. (c) Thepchatri,
P.; Cicero, D. O.; Monteagudo, E.; Ghosh, A. K.; Cornett, B.; Snyder,
J. P. Conformations of Laulimalide in DMSO-d6. J. Am. Chem. Soc.
2005, 127, 12838-12846.
(58) (a) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.;
Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shaw, D.
E.; Shelley, M.; Perry, J. K.; Sander, L. C.; Shenkin, P. S. Glide: A
New Approach for Rapid, Accurate Docking and Scoring. 1. Method
and Assessment of Docking Accuracy. J. Med. Chem. 2004, 47,
1739-1749. (b) Halgren, T. A.; Murphy, R. B.; Friesner, R. A.;
Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. Glide: A
New Approach for Rapid, Accurate Docking and Scoring. 2.
Enrichment Factors in Database Screening. J. Med. Chem. 2004, 47,
1750-1759.
(59) Louie, K. G.; Behrens, B. C,; Kinsella, T. J.; Hamilton, T. C.;
Grotzinger, K. R.; McKoy, W. M.; Winker, M. A.; Ozols, R. F.
Radiation Survival Parameters of Antineoplastic Drug-sensitive and
Resistant Human Ovarian Cancer Cell Lines and Their Modification
by Buthionine Sulfoximine. Cancer Res. 1985, 45, 2110-2115.
(60) Chang, M. C.; Uang, B. J.; Wu, H. L.; Lee, J. J.; Hahn, L. J.; Jeng,
J. H. Inducing The Cell Cycle Arrest and Apoptosis of Oral KB
Carcinoma Cells by Hydroxychavicol: Roles of Glutathione and
Reactive Oxygen Species. Br. J. Pharmacol. 2002, 135, 619-630.
(61) (a) Chatterjee, S. K.; Barron, D. M.; Vos, S.; Bane, S. Baccatin III
Induces Tubulin to Assemble into Long Microtubules Biochemistry
2001, 40, 6964-6970. (b) Barron, D. M.; Chatterjee, S. K.; Ravindra,
R.; Roof, R.; Baloglu, E.; Kingston, D. G. I.; Bane, S. A Fluorescence-
Based High-Throughput Assay for Antimicrotubule Drugs. Anal.
Biochem. 2003, 315, 49-56.
(45) For leading references, see: Littke, A. F.; Schwarz, L.; Fu, G. C.
Pd/P(t-Bu3): A Mild and General Catalyst for Stille Reactions of
Aryl Chlorides and Aryl Bromides. J. Am. Chem. Soc. 2002, 124,
6343-6348, and references cited therein.
(46) Chen, S.-H.; Kadow, J. F.; Farina, V.; Fairchild, C. R.; Johnston, K.
A. First Synthesis of Novel Paclitaxel (Taxol) Analogues Modified
at the C4-Position. J. Org. Chem. 1994, 59, 6156-6158.
(47) Holton, R. A.; Zhang, Z.; Clarke, P. A.; Nadizadeh, H.; Procter, D.
J. Selective Protection of the C(7) and C(10) Hydroxyl Groups in
10-Deacetyl Baccatin III. Tetrahedron Lett. 1998, 39, 2883-2886.
(48) (a) Holton, R. A, Biediger, R. J.; Boatman, P. D. Semisynthesis of
Taxol and Taxotere. In Taxol: Science and Application; Suffness,
M.; Ed.; CRC Press: New York, 1995; pp 97-121. (b) Georg, G.
I.; Boge, T. C.; Cheruvallath, Z. S.; Clowers, J. S.; Harriman, G. C.
B.; Hepperle, M.; Park, H. The Medicinal Chemistry of Taxol. In
Taxol: Science and Application; Suffness, M., Ed.; CRC Press: New
York, 1995; pp 317-375. (c) Ojima, I.; Habus, I.; Zhao, M.; Zucco,
M.; Park, Y. H.; Sun, C. M.; Brigaud, T. New and Efficient
Approaches to the Semisynthesis of Taxol and its C-13 Side Chain
Analogs by Means of â-Lactam Synthon Method. Tetrahedron 1992,
48, 6985-7012. (d) Ojima, I.; Lin, S.; Chakravarty, S.; Fenoglio, I.;
Park, Y. H.; Sun, C-M.; Appendino, G.; Pera, P.; Veith, J. M.;
Bernacki, R. J. Syntheses and Structure-Activity Relationships of
Novel Nor-seco Taxoids. J. Org. Chem. 1998, 63, 1637-1645.
(49) The reader should note that Phe272 is the pdb structure code for the
Phe 270 sequence code (Fâ270V) in Table 2.
(50) Wang, M.; Xia, X.; Kim, Y.; Hwang, D.; Jansen, J. M.; Botta, M.;
Liotta, D. C.; Snyder, J. P. A Unified and Quantitative Receptor
JM061071X