Design and synthesis of (+)-discodermolide-paclitaxel hybrids leading to enhanced biological activity

Article abstract of DOI:10.1021/jm200692n

Potential binding modes of (+)-discodermolide at the paclitaxel binding site of tubulin have been identified by computational studies based on earlier structural and SAR data. Examination of the prospective binding modes reveal that the aromatic pocket oc

Full text of DOI:10.1021/jm200692n

ARTICLE
pubs.acs.org/jmc
Design and Synthesis of (+)-DiscodermolideꢀPaclitaxel Hybrids
Leading to Enhanced Biological Activity
Amos B. Smith, III,*,† Keizo Sugasawa,†,§ Onur Atasoylu,† Chia-Ping Huang Yang,‡ and
Susan Band Horwitz*,‡
†Department of Chemistry, Monell Chemical Senses Center and Laboratory for Research on the Structure of Matter,
University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
‡Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, United States
§Astellas Pharma Inc., Tsukuba-shi, Ibaraki 305-8585, Japan
S Supporting Information
b
ABSTRACT:
Potential binding modes of (+)-discodermolide at the paclitaxel binding site of tubulin have been identified by computational
studies based on earlier structural and SAR data. Examination of the prospective binding modes reveal that the aromatic pocket
occupied by the paclitaxel side chain is unoccupied by (+)-discodermolide. Based on these findings, a small library of (+)-
discodermolideꢀpaclitaxel hybrids have been designed and synthesized. Biological evaluation reveals a two- to eight-fold increase in
antiproliferative activity compared to the parent molecule using the A549 and MCF-7 cancer cell lines.
’ INTRODUCTION
to access the C14ꢀC15 olefin.7e,9 Subsequently, Novartis em-
ployed a similar cross-coupling tactic (i.e., Suzuki) to provide
(+)-discodermolide for their phase I clinical trials.10 Also early
on, we also proposed11 a solution conformation of (+)-disco-
dermolide based on a combination of NMR data and the solid-
state X-ray structure and carried out a comprehensive structureꢀ
activity relationship (SAR) study with Kosan Biosciences,
Inc.12,13
More recently, we demonstrated that (+)-discodermolide
both causes accelerated cell senescence14 and binds to a region
on β-tubulin close to the paclitaxel binding site.15 Of interest
here, (+)-discodermolide and paclitaxel act synergistically, not
only in cell culture16 but also in an ovarian xenograft tumor
model in nude mice.17 Subsequent studies, utilizing hydrogen/
deuterium exchange and mass spectrometry, indicated that
paclitaxel and discodermolide induce stability on opposite sides
of the microtubule interface, suggesting a possible explanation
for the synergy observed between these two drugs.18 Finally, to
(+)-Discodermolide [(+)-1], a potent antitumor polyketide
natural product, was first isolated in the early 1990s from
extracts of the Caribbean marine sponge Discodermia dissoluta
by Gunasekera and co-workers.1 Confirmation of the assigned
structure, as well as the absolute stereochemistry, was subse-
quently achieved by Schreiber and co-workers via total syntheses
of the (ꢀ) enantiomer (1993), the natural (+) enantiomer
(1994), and a number of novel analogues.2,3 Initially reported
to be a potent immunosuppressive agent, (+)-discodermolide
was later recognized to possess significant antiproliferative
activity both in vitro4a across the NCI panel of human cancer
cell lines,5 including paclitaxel resistant cell lines,6 and in vivo.4b
Like paclitaxel (2), (+)-discodermolide stabilizes tubulin thereby
modulating microtubule dynamics that leads to cell arrest at
mitosis (Figure 1).
As a result of the observed antitumor activity, (+)-discoder-
molide has generated significant interest in the synthetic commu-
nity: 13 total syntheses have been reported to date.7 Of particular
significance, as recently recognized,8 our four generation strate-
gies exploited an early example of the Negishi coupling protocol
Received: May 31, 2011
Published: August 26, 2011
r
2011 American Chemical Society
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structure,1 notwithstanding the 15 rotatable σ bonds.11 In
particular, variable temperature and cosolvent titration NMR
studies provided evidence of intramolecular hydrogen bonds
between the C(7)-OH and the C(1)-carbonyl, in conjunction
with interactions between the carbamate nitrogen and the C(3)
and C(7) hydroxyl groups. We postulated that syn-pentane
interactions21 were responsible for the proposed turn geometry
of the backbone. Importantly, biological studies of epimers
bearing inverted stereogenicities at the C(7), C(11), C(16),
and C(17) revealed significantly lower activities, which led us
to suggest that the conformation of the molecular segment
between C(7)ꢀC(17) is conserved upon binding. These con-
clusions were later confirmed by studies from other research
laboratories.20
Figure 1. (+)-Discodermolide and paclitaxel.
To understand more fully the solution behavior of (+)-
discodermolide, we recently initiated computational studies
exploiting molecular dynamics (MD) simulations. Monitoring
backbone torsional angles over a 1 μs simulation period in water
revealed the flexible torsional angles. It is of considerable interest
that the lactone ring and the diene side chain moieties were found
to display more flexibility compared to the rest of the molecule,
when the turn geometry [i.e., C(7)ꢀC(16)] was maintained
throughout the simulation.
decrease the pneumotoxicity of (+)-1, the presumptive cause
leading to the failure of the Novartis phase I clinical trial,10
possibly due to metabolites,19 we extended our analogue pro-
gram wherein the metabolically less stable lactone portion of the
molecule13aꢀc and C14ꢀC15 olefin13d were either individually or
together replaced with isosteres. Pleasingly, these analogues pre-
served the cytotoxicity at a level similar to (+)-discodermolide (1).
However, despite the availability of extensive SAR data,12 as
well as structural,1 computational and NMR studies,11,20 the
binding mode of (+)-discodermolide on tubulin remains unclear.
Toward this end, we report here two possible binding modes of
(+)-discodermolide in conjunction with the design, synthesis,
and biological evaluation of (+)-discodermolide/paclitaxel
hybrid molecules possessing photoaffinity labels.
In our SAR studies, we demonstrated the following: (1) that
changes in the substitution pattern of C(1), C(3), and C(7)
centers of (+)-discodermolide are favorable to retention of
potent cytotoxicity, (2) that variations at the C(2), C(14), and
the carbamate carbons are tolerable, and (3) that changes within
the remaining portions of (+)-discodermolide significantly
decrease the cytotoxicity.12,13 On the basis of these observations,
in conjunction with our earlier photoaffinity labeling study,13e,f
we reasoned that the diene terminus of discodermolide resides in
a hydrophobic/aromatic pocket, while the lactone moiety either
occupies a binding pocket that can accommodate large groups or,
more likely, is partially interfaced with the solvent. Further
information gained from our earlier photoaffinity labeling study
revealed that attachment of a large aromatic system (cf. ben-
zophenone) and/or alkyl groups to the carbamate nitrogen did
not significantly alter the observed activity.13f In fact, in two cases,
comprising attachment of a p-dimethylaniline or p-benzophe-
none substituent at the carbamate nitrogen, a significant increase
in tumor cell growth inhibitory activity was observed. We
emphasize, however, that our earlier study was directed specifi-
cally at the preparation of photoaffinity labeled (+)-discodermo-
lide analogues, with little or no attention focused on activity
changes except for the possibility of increased hydrophobicity
and/or conformational preferences. A closer examination of the
earlier carbamate analogues, however, reveals a strong activity
dependency both on the nature of the aromatic rings and
linker length. On the basis of these observations, we have now
examined the scope and molecular basis for the observed increase
in cytotoxicity of these analogues exploiting detailed computa-
tional analysis.
We next turned to NMR experiments to refine the solution
conformation. The Distribution of Solution Conformations
(DISCON) program developed in our laboratory22 was
employed to obtain a distribution of conformers based on NMR
data. Earlier studies had revealed that (+)-discodermolide is more
flexible in organic solvents.20a,b On the basis of this observation,
acetonitrile was selected as the solvent for the NMR experiments,
given that the aim of this study was to identify the “flexible”
torsional angles and possible diverse conformational families for
our pending docking studies. Protonꢀproton coupling constants
and NOE derived distances reported earlier were utilized.11
Deconvolution of the NMR observables over an ensemble of
structures obtained by Monte Carlo conformational searches,
followed by the clustering analysis that is embedded in the
DISCON software, led to an ensemble of four principal con-
formers that in combination fit the NMR derived distance and
torsional angle data better than any single conformation. Two
conformers (Figure 2A,B), similar to the solid-state structure
with differences in the lactone ring orientation, were identified as
the major solution conformations (A, 35%; B, 32%), an observa-
tion fully consistent with our earlier solution structure.11 Further
examination of members of the major conformational families
revealed (+)-discodermolide can adopt different conformations
by adjusting the torsional angles around C(11)ꢀC(12) and C(7)
and C(8) bonds. In addition, the lactone ring can exist in equi-
librium between chair, half-chair, and skew-boat conformations.
The next most populated conformer (Figure 2C, 26%) com-
prised an inverted orientation at the diene region, wherein a dif-
ferent gauche orientation is preferred around the C(16)ꢀC(17)
and C(18)ꢀC(19) bonds, with the remaining regions of (+)-
discodermolide identical to the solid state structure. The remain-
ing major family (Figure 2D, 7%) comprises both of these con-
formational changes and thereby forces the molecule to occupy
an extended conformation. The distribution of the conformations
3
3
can be rationalized by the observed JH16ꢀH17 and JH11ꢀH12
values of 6.2 and 6.6 Hz, respectively, corresponding to torsional
angle averaging and by the observed NOE correlation variations
from the solid-state structure, in particular those between H(7)ꢀ
H(9), H(8)ꢀH(12), H(9)ꢀH(11), and H(12)ꢀMe(18). Similar
The Solution Conformations of (+)-Discodermolide. In
2001, we reported that the preferred major solution conforma-
tion of (+)-discodermolide (1) was similar to the solid-state
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(+)-discodermolide is treated as a fully flexible molecule without
regard to the accessible solution conformations.15,18,30 Finally,
earlier docking studies did not take into account the reported
SAR data,19 in particular the potent carbamate analogues.13f
More recently, Carlomagno20a and then Jimꢀenez-Barbero
et al.20b concluded, based on NMR transfer NOE studies of
(+)-discodermolide and tubulin, that the binding conformation
of (+)-discodermolide is identical to the solid state conforma-
tion with differences in the lactone ring portion and that
(+)-discodermolide binds at the paclitaxel binding site. Paterson
and co-workers in turn, based on these proposals, prepared a
series of discodermolideꢀdictyostatinꢀpaclitaxel hybrids which
upon biological evaluation revealed a 20ꢀ35-fold decrease in
tumor cell growth inhibition activity, employing the PANC-1
human cancer cell lines.31 Notwithstanding these disappointing
observations, we undertook docking studies employing the
solution conformations identified by DISCON (vide infra).
Our 2009 hydrogenꢀdeuterium exchange experiments also
revealed that (+)-discodermolide binds to a region quite close to
the paclitaxel site on β-tubulin, however, the stabilized micro-
tubule geometries were different, as evidenced by the observed
solvent exposure of the lateral sites,18 with the most important
interactions observed in the M-loop region of β-tubulin. On the
basis of this information, we also chose to employ the paclitaxel
site for docking studies. Beginning with the paclitaxel bound
protein structure (PDB: 1JFF), paclitaxel was removed and the
remaining protein structure was minimized with the OPLS-2005
force field to remove possible steric clashes. The binding site was
not altered excessively, as we did not want to dock to a
computationally generated binding pocket. Sets of (+)-discoder-
molide conformations, belonging to the families identified in
solution (Figure 2AꢀD), were then docked, employing the
Glide32 and Molegro33 software packages, treating the (+)-
discodermolide conformations as rigid structures and the protein
structure as flexible. Docking modes inconsistent with the SAR
data were eliminated by visual inspection. We reason that this
approach provides more valid criteria for finding a working
binding hypothesis than calculated docking scores and energies
due to the uncertainties (vide infra) in the protein structure
(cf. Supporting Information for elimination criteria). Two bind-
ing poses for (+)-discodermolide were identified to be consistent
with the SAR data.
The first pose (Figure 3B) orients the lactone end of (+)-
discodermolide in a region occupied by the baccatin core of
paclitaxel. The turn structure of (+)-discodermolide fits tightly in
a region under the M-loop (β212ꢀ230), with the C(3) and C(7)
hydroxyl groups forming hydrogen bonds to Pro272 and
Thr274. In this pose, the carbamate moiety is directed toward
the aromatic pocket that accommodates the paclitaxel tail
(Figure 3A). The diene side chain and the lactone carbonyl
group are directed toward the solvent, where additional substit-
uents would appear to be tolerated. Rescoring of this pose via the
MM/GBSA protocol34 provided a favorable ΔG value of ꢀ12
kcal/mol. The second pose (Figure 3C), similar to pose 1, utilizes
the (+)-discodermolide turn scaffold to fit in the hydrophobic
pocket under the M-loop. This pose, however, orients the lactone
moiety to the region where the paclitaxel C(2) benzoate group
binds. This region would also appear to be able to accommodate
larger groups while maintaining the activity of (+)-discodermo-
lide analogues. That is, replacement of the lactone ring with other
moieties might prove feasible. In this pose, the known decrease in
activity of (+)-discodermolide analogues upon acetylation at the
Figure 2. Solution conformations of (+)-discodermolide.
analyses reported earlier by the Jimꢀenez-Barbero20b and the Snyder
Laboratories20c are in accord with these observations, wherein
differences in distributions were observed, presumably due to the
differences in the solvents employed in the NMR experiments as
well as to the force fields and clustering methods employed.
In summary, (+)-discodermolide in solution displays consid-
erable flexibility in the orientation of the lactone and the diene/
carbamate termini, with conservation of the turn region of the
conformation comprising the C(7)ꢀC(16) centers. For docking
studies, we reasoned that the solution structures hold consider-
able importance because the bioactive conformation is more
likely to exist in solution given that a lower energy penalty is paid
for ligand reorganization upon binding (i.e., preorganization).23
Stated differently, the 3D binding pharmacophore of the ligand is
likely to be found in the conformation(s) that exist in solution,
thus permitting easy recognition.24
Docking Studies. Having an understanding of the available
major ensemble of solution conformations, we turned to define
the binding mode of (+)-discodermolide on microtubules. In
general, docking studies of microtubule stabilizing agents utilize
the heterodimer Rꢀβ tubulin structure (PDB code: 1JFF)
obtained by high resolution electron microscopy from the Zn
induced crystalline sheets of tubulin in the presence of
paclitaxel.25 Although the tubulin structure in the crystal was
shown to match the microtubule structure, it remains only an
approximate model for docking studies for the following reasons:
(1) the protein structure is distorted by the bound paclitaxel and
thus does not necessarily represent the unbound microtubule,26
(2) in vivo orientations and conformations of tubulin in micro-
tubules and Zn-induced tubulin crystal sheets are different,27 (3)
the flexible M-loop region, at the taxane binding site is not well
resolved,25 (4) the lateral contacts between protofilaments,
which are important for microtubule formation and tubulin
dynamics28 are missing, and (5) in humans the tubulin isotypes
have different sequences than found in the reported protein
structure.29 Equally significant, the majority of reported (+)-
discodermolide docking studies employ either the solid state
conformation of (+)-discodermolide as a rigid scaffold or
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poses presented here and was found to be inconsistent with our
SAR criteria.
Design of Hybrid Analogues. On the basis of the (+)-
discodermolide docking modes, we designed a series of analo-
gues that we reasoned would hold the promise of not only
confirming our binding hypothesis, based initially on enhanced
binding to tubulin and in turn enhanced cytotoxicity, but also
define the true binding pose. Importantly, the design criteria
were orchestrated to accommodate incorporation of photoaffi-
nity labels for future structural studies.
Utilizing docking poses 1 and 2, we envisioned filling the
aromatic pocket in the vicinity of the carbamate group, which in
the case of paclitaxel is occupied by C(30)-Ph group. In silico
docking of our earlier carbamate analogues (vide infra), employ-
ing the identified (+)-discodermolide poses, revealed that the
earlier analogues did not fully occupy the proposed pocket due to
the shorter tether lengths. To test the validity of this scenario,
analogues having aromatic groups attached to the carbamate
group with differing tether lengths were generated (Figure 4).
Figure 4. Designed (+)-discodermolide analogues.
We hypothesized that if the binding pocket is valid, the
cytotoxic activities should vary, with the analogue possessing
the most suitable linker length displaying the highest tumor cell
growth inhibition activity. A second amide linkage was incorpo-
rated onto the tether to participate in a possible hydrogen bond,
with Asp26 and His229 of β-tubulin, similar to the paclitaxel
amide linkage. Finally, we envisioned that photoaffinity tags
could be appropriately introduced without deleterious effects on
the cytotoxicity. To this end, (+)-discodermolide/paclitaxel hybrid
structures with a p-azidophenyl substituent (3ꢀ5, Figure 4) were
generated. Docking studies were then conducted similar to the
procedure applied for (+)-discodermolide. In these studies, the
newly introduced paclitaxel C(30)-phenyl group surrogates were
permitted to be flexible, whereas the (+)-discodermolide core
was kept rigid. Examination of the docking scores identified
hybrid 4 to fill the aromatic pocket (Figure 5). Two additional
hybrids 6 and 7 possessing different photoaffinity tags and optimal
linker lengths were also designed to explore the potential effects
of the hybrid structure motif as well as the photoaffinity groups
on the cytotoxicity.
Figure 3. Binding mode of (A) paclitaxel (PDB: 1JFF), (B) (+)-
discodermolide/pose 1, (C) (+)-discodermolide/pose 2.
C(11) hydroxyl group can be rationalized by the loss of a
hydrogen bond to Thr274. This pose also orients the diene side
chain in line with the paclitaxel benzamide group, with the (+)-
discodermolide carbamate group directed to the C(30)-phenyl
pocket. Rescoring employing the MM/GBSA protocol again
provided a favorable ΔG value of ꢀ18 kcal/mol. It is noteworthy
that the Jimꢀenez-binding mode differed significantly from the
Synthesis of the Designed Analogues. The requisite
hybrids, carrying the photoaffinity tags, were envisioned to
arise via union of known (+)-discodermolide intermediate 19
(Scheme 1),7e prepared in connection with our gram-scale
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Scheme 1. Retrosynthetic Analysis of Hybrid Molecules
Scheme 2. Preparation of the Photoaffinity Tags
Figure 5. Docking of 4 in (A) pose 1 and (B) pose 2.
Scheme 3. Syntheses of the Designed Analogues
synthesis,11 with the photoaffinity units bearing the suitable
amine linkers. One of the attributes of developing preparative
scale synthesis of architecturally complex natural products pos-
sessing significant bioregulatory properties is the subsequent
ready availability of advanced intermediates for SAR and photo-
affinity labeling studies.
To permit facile formation of carbamate linkage, the secondary
alcohol on (+)-discodermolide would be activated as a carbonate
ester (cf. 20). The amine coupling partners were envisioned to
arise via condensation reactions of the photoaffinity tags posses-
sing carboxylic acid functionality with the diamine tethers of
suitable length.
The photoaffinity tags for this study were prepared following
known synthetic routes,35ꢀ37 wherein acids 8 and 13 were
activated as hydroxysuccinamide esters,38 followed by condensa-
tion with the alkyl diamine linkers (Scheme 2). For the aryl-(tri-
fluoromethyl)diazirine component 18, the mono-Boc protected
amine linker was employed with N,N0-carbonyl-diimidazole
(CDI)39 to reduce possible side reactions; the Boc group was
subsequently removed under acidic conditions.
two-step protocol wherein the secondary alcohol in (+)-19 was
converted to a p-nitrophenylcarbonate ester, followed by union
In the event, construction of (+)-discodermolide/paclitaxel
hybrids 3ꢀ7 (Scheme 3), possessing the phototags, entailed a
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Table 1. Cytotoxicity of (+)-Discodermolide and Analogues against Human Lung A549 and Breast MCF-7 Cancer Cell Lines
a
IC50
(+)-1
(+)-2
(+)-3
(+)-4
(+)-5
(+)-6
(+)-7
A549
9.34 ( 0.56
7.98 ( 0.35
3.14 ( 0.09
0.91 ( 0.11
5.61 ( 0.15
3.64 ( 0.12
1.21 ( 0.35
1.90 ( 0.43
3.76 ( 0.57
2.71 ( 0.55
1.05 ( 0.15
1.66 ( 0.48
2.64 ( 0.22
1.92 ( 0.07
MCF-7
a IC50 equals the concentration (nM) of drug that inhibits 50% cell growth.
with amines 10ꢀ12 and 15 and 18.40 Global deprotection under
acidic conditions provided (+)-3ꢀ(+)-7 in good to excellent
yields. Importantly, the synthetic route not only provides facile
access to the designed (+)-discodermolide/paclitaxel hybrids
possessing the photoaffinity tags, but was also designed to permit
’ EXPERIMENTAL SECTION
Experimental Details for Obtaining IC50 Values. Cells lines
were obtained from the American Type Culture Collection (ATCC)
and maintained in RPMI medium supplemental with 10% fetal bovine
serum. Cells (800 A549 or 2000 MCF-7) were added to each well of a
96-well plate. Increasing concentrations of the indicated drugs were
added 18 h after plating. IC50 values, the concentration of drug that
inhibits cell growth by 50%, were determined after 72 or 96 h incubation
at 37 °C for A549 or MCF-7 cells, respectively, using the SRB method.40
The two cell lines were fixed and stained at different times to take into
account their different growth rates.
3
ready access to either H or 14C analogues for future labeling
experiments (Scheme 3).
Biological Evaluations of the (+)-Discodermolide/Pacli-
taxel Hybrids. The designed (+)-discodermolide/paclitaxel
hybrids possessing the photoaffinity labels were tested for
antiproliferative activity against human lung (A549) and breast
(MCF-7) cancer cell lines. Discodermolide [(+)ꢀ1] and paclitaxel
(2) served as controls. In accord with our design strategy, the
analogues revealed improved activity in a trend changing with the
tether length (Table 1).
Given the similarity of the analogues, the up to an 8-fold
increase in potency is believed to be associated with an interac-
tion with the proposed aromatic pocket and not simply to an
increase in hydrophobicity or other factors. Also of importance,
(+)-discodermolide and each of the six analogues increased the
amount of microtubule polymerization compared to the control
that had no drug added (Figure 6). Taken together, these
observations strongly suggest that all of the discodermolide
molecules have the same mechanism of action.
Experimental Details for Tubulin Polymerization Studies.
Bovine brain tubulin (2.5 μM) from Cytoskeleton Inc. in a buffer
containing 0.1 M MES, 1 mM EGTA, and 1 mM MgCl2 was incubated
with 3 μM of the indicated drugs, in the presence of 2 mM GTP, at 37 °C
for 30 min. Samples were centrifuged at 120000g at 37 °C for 1 h, and the
pellet that contains the polymerized tubulin was dissolved in SDS sample
buffer and analyzed by SDS-polyacrylamide gel electrophoresis. Proteins
were then transferred to nitrocellulose and stained with Ponceau S.
Experimental Details for Synthesis and Characterization.
Except as otherwise indicated, all reactions were run under an argon
atmosphere in flame- or oven-dried glassware, and solvents were freshly
distilled. The argon was deoxygenated and dried by passage through an
OXICLEAR filter from Aldrich and Drierite tube, respectively. Diethyl
ether (Et2O), tetrahydrofuran (THF), and dichloromethane (CH2Cl2)
were purchased from Aldrich (HPLC purity) and further purified by
Pure Solve PS-400. All other reagents were purchased from Aldrich or
Acros and used as received. Reactions were monitored by thin layer
chromatography (TLC) either with 0.25 mm of Silicycle or 0.25 mm of
E. Merck (Kieselgel 60F254, Merck) precoated silica gel plates. Silica gel
for flash chromatography (particle size 0.040ꢀ0.063 mm) was supplied
by Silicycle or Sorbent. Yields refer to chromatographically and spectro-
scopically pure compounds unless otherwise noted. 1H and 13C spectra
were recorded on a Bruker AMX-500 spectrometer. Chemical shifts are
reported as δ values relative to internal chloroform (δ 7.26) or benzene
(δ 7.15) for 1H and either chloroform (δ 77.0) or benzene (δ 128.0) for
13C. Infrared spectra were recorded on a Jasco FTIR-480plus spectro-
meter. Optical rotations were measured on a Jasco P-2000 polarimeter in
the solvent indicated. High resolution mass spectra were measured at the
University of Pennsylvania Mass Spectrometry Center on either a VG
Micromass 70/70H or VG ZAB-E spectrometer. Analytical reverse-
phased (Sunfire C18; 4.6 mm ꢁ 50 mm, 5 mL) high-performance liquid
chromatography (HPLC) was performed with a Waters binary gradient
module 2525 equipped with Waters 2996 PDA and Waters micromass
ZQ. All final compounds were analyzed employing a linear gradient from
10% to 90% of acetonitrile in water over 8 min and a flow rate of 1 mL/
min and, unless otherwise stated, the purity level was >95%.
Figure 6. Tubulin polymerization in the presence of (+)-discodermo-
lide and its analogues. The control has no drug.
In summary, we have defined two possible binding modes for
(+)-discodermolide in the paclitaxel binding pocket. To test the
validity of the proposed modes, we designed and synthesized a
series of hybrid molecules wherein the aromatic ring of paclitaxel
was attached to (+)-discodermolide, exploiting a tether of
suitable length. Biological testing demonstrated that hybrid
congeners possess increased potency compared to (+)-discoder-
molide. Given the availability of the already incorporated photo-
tags, the discodermolide/paclitaxel hybrids hold the promise of
defining the (+)-discodermolide binding site and mode of action,
as well as determining whether the drug demonstrates selectivity
for any of the 7 β-tubulin isotypes. Because certain isotypes,
particularly βIII-tubulin, have been associated with paclitaxel
resistance,41 an investigation of the interaction between drugs
and specific tubulin isotypes becomes an important goal, and in
particular definition of human tubulin isotypes that bind (+)-
discodermolide. Such studies in conjunction with radiolabeling
experiments are ongoing in our laboratories.
Representative Synthetic Procedures. Synthesis of (+)-3a.
Compound (+)-20 (80 mg, 0.0725 mmol) was dissolved in CH2Cl2
(1 mL) and MeOH (1 mL). N-(4-Azido-benzoyl)-1,2-ethanediamine
(10) (45 mg, 0.218 mmol) in MeOH (1 mL) and Et3N (40 μL,
0.290 mmol) were added to the above solution. The resultant solution
was stirred at room temperature for 6 days. Water (50 mL) was added to
the reaction mixture and extracted with CH2Cl2 (3 ꢁ 50 mL). The
combined organic layers were washed with brine (50 mL), dried over
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MgSO4, filtered, and concentrated. Flash chromatography (11ꢀ25%
EtOAc in hexane) provided compound (+)-3a (75.1 mg, 89% yield) as a
colorless amorphous solid. [R]22D = +36.0° (c = 0.65, CHCl3). IR (film,
NaCl): 3336, 2957, 2929, 2857, 2123, 1718, 1653, 1604, 1539, 1500,
1254, 1044, 836, 775 cmꢀ1. 1H NMR (500 MHz, CDCl3) δ: 7.81 (d, J =
8.3 Hz, 2H), 7.33 (brs, 1H), 7.03 (d, J = 8.4 Hz, 2H), 6.54 (ddd, J = 16.7,
10.6, 10.6 Hz, 1H), 5.88 (apparent t, J = 10.9 Hz, 1H), 5.35ꢀ5.13 (m,
5H), 5.11 (d, J = 10.2 Hz, 1H), 5.02 (d, J = 9.9 Hz, 1H), 4.81 (apparent t,
J = 9.1 Hz, 1H), 4.74 (apparent t, J = 5.8 Hz, 1H), 4.59 (ABq, JAB = 6.9
Hz, ΔAB = 28.4 Hz, 2H), 4.50 (apparent t, J = 10.3 Hz, 1H), 3.63 (brs,
1H), 3.56ꢀ3.44 (m, 3H), 3.41 (apparent t, J = 4.0 Hz, 1H), 3.39ꢀ3.30
(m, 1H), 3.34 (s, 3H), 3.06 (apparent t, J = 5.6 Hz, 1H), 2.99ꢀ2.91 (m,
1H), 2.75ꢀ2.66 (m, 1H), 2.60 (qd, J = 7.5, 2.8 Hz, 1H), 2.55ꢀ2.47 (m,
1H), 2.06 (apparent t, J = 12.7 Hz, 1H), 1.91ꢀ1.76 (m, 3H), 1.75ꢀ1.66
(m, 2H), 1.63ꢀ1.54 (m, 1H), 1.57 (s, 3H), 1.23 (d, J = 7.6 Hz, 3H), 0.96
(d, J = 6.7 Hz, 3H), 0.95ꢀ0.84 (m, 12H), 0.91 (s, 9H), 0.87 (s, 9H), 0.85
(s, 9H), 0.72 (d, J = 6.7 Hz, 3H), 0.08 (s, 3H), 0.06 (s, 3H), 0.054
(s, 3H), 0.048 (s, 3H), 0.04 (s, 3H), 0.03 (s, 3H). 13C NMR (125 MHz,
CDCl3) δ: 173.6, 166.7, 158.8, 143.4, 133.9, 133.4, 132.3, 132.1, 132.0,
131.1, 130.8, 130.0, 129.0, 119.1, 118.5, 97.5, 86.4, 79.5, 77.3, 77.0, 74.9,
64.9, 56.2, 44.2, 42.7, 42.5, 40.5, 37.9, 36.1, 35.8, 35.7, 34.6, 34.4, 34.3,
26.4, 26.1, 25.9, 23.3, 18.7, 18.3, 18.1, 17.7, 16.8, 16.7, 16.5, 14.4, 14.2,
10.2, ꢀ3.2, ꢀ3.5, ꢀ4.2, ꢀ4.3, ꢀ4.66, ꢀ4.68. High resolution mass
spectrum (ESI+) m/z 1190.7408 ((M + Na)+; calcd for C62H109N5O10-
Si3Na: 1190.7380).
in computational studies. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For A.B.S.: phone, (215) 898-4860; fax, (215) 898-5129;
E-mail, smithab@sas.upenn.edu. For S.B.H.: phone, (718) 430-
2163; fax, (718) 430-8922; E-mail, susan.horwitz@einstein.yu.edu.
’ ACKNOWLEDGMENT
Support was provided by the National Institutes of Health
through the National Institutes of General Medical Sciences and
the National Cancer Institute through grants GM-29028 (A.B.S.)
and CA-077263 (S.B.H.), and the Breast Cancer Research
Foundation (S.B.H.). We also thank Drs. G. Furst and R. Kohli
of the Department of Chemistry NMR and MS Facilities for
obtaining the NMR and high resolution mass spectra.
’ ABBREVIATIONS USED
ESI-HMRS, electrospray ionization high-resolution mass spec-
trometry; TMS, tetramethylsilane; TLC, thin layer chromatog-
raphy; PDB, Protein Data Bank; DISCON: distribution of
solution conformationsNCI, National Cancer Institute; SAR,
structureꢀactivity relationship; MD, molecular dynamics; CDI,
N,N0-carbonyl-diimidazole
Synthesis of (+)-3. To a solution of compound (+)-3a (67 mg,
0.0573 mmol) in MeOH (5 mL) was added aqueous hydrochloric
acid (4M, 4 mL) in 100ꢀ250 μL portions over 7 h at a rate which
minimized precipitation (ca. 8ꢀ30 min intervals), and the sides of flask
were rinsed with MeOH (1 mL). The reaction mixture was stirred at
room temperature for 18 h and diluted with EtOAc (70 mL). The
resulting solution was neutralized with NaHCO3 (1.35 g in H2O 18 mL)
at 0 °C. Phosphate buffer pH 7 (1M, 30 mL) and NaCl (17 g) were
added, and the aqueous layer was extracted with EtOAc (3 ꢁ 70 mL).
The combined organic layers were washed with brine (100 mL), dried
over MgSO4, filtered, and concentrated. The residue was purified three
times by flash chromatography (80% EtOAc in hexane or 3ꢀ6%
MeOH/CH2Cl2) to afford compound (+)-3 (31.1 mg, 69% yield) as
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D
(film, NaCl): 3359, 2968, 2123, 1699, 1639, 1604, 1543, 1501, 1457,
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’ ASSOCIATED CONTENT
1
Supporting Information. H and 13C NMR spectra, experi-
S
b
mental methods and synthetic procedures of (+)-3ꢀ(+)-20;
HPLC data of (+)-3ꢀ(+)-7 used in biological assays; NMR data
utilized in solution conformation calculations; SAR data employed
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