Design, synthesis, and biological evaluation of diminutive forms of (+)-spongistatin 1: Lessons learned

Article abstract of DOI:10.1021/ja2046167

The design, synthesis, and biological evaluation of two diminutive forms of (+)-spongistatin 1, in conjunction with the development of a potentially general design strategy to simplify highly flexible macrocyclic molecules while maintaining biological activity, have been achieved. Examination of the solution conformations of (+)-spongistatin 1 revealed a common conformational preference along the western perimeter comprising the ABEF rings. Exploiting the hypothesis that the small-molecule recognition/binding domains are likely to comprise the conformationally less mobile portions of a ligand led to the design of analogues, incorporating tethers (blue) in place of the CD and the ABCD components of the (+)-spongistatin 1 macrolide, such that the conformation of the retained (+)-spongistatin 1 skeleton would mimic the assigned solution conformations of the natural product. The observed nanomolar cytotoxicity and microtubule destabilizing activity of the ABEF analogue provide support for both the assigned solution conformation of (+)-spongistatin 1 and the validity of the design strategy.

Full text of DOI:10.1021/ja2046167

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pubs.acs.org/JACS
Design, Synthesis, and Biological Evaluation of Diminutive Forms
of (+)-Spongistatin 1: Lessons Learned
Amos B. Smith, III,*,† Christina A. Risatti,†,‡ Onur Atasoylu,† Clay S. Bennett,†,# Junke Liu,§
Hongsheng Cheng,§ Karen TenDyke,§ and Qunli Xu^
†Monell Chemical Senses Center and Laboratory for Research on the Structure of Matter, Department of Chemistry,
University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
§Next Generation Systems and ^Oncology Product Creation Unit, Eisai, Inc., Andover, Massachusetts 01810, United States
S Supporting Information
b
ABSTRACT: The design, synthesis, and biological evaluation
of two diminutive forms of (+)-spongistatin 1, in conjunction
with the development of a potentially general design strategy to
simplify highly flexible macrocyclic molecules while maintaining
biological activity, have been achieved. Examination of the
solution conformations of (+)-spongistatin 1 revealed a com-
mon conformational preference along the western perimeter
comprising the ABEF rings. Exploiting the hypothesis that the
small-molecule recognition/binding domains are likely to com-
prise the conformationally less mobile portions of a ligand led to
the design of analogues, incorporating tethers (blue) in place of
the CD and the ABCD components of the (+)-spongistatin 1
macrolide, such that the conformation of the retained (+)-spongistatin 1 skeleton would mimic the assigned solution conformations
of the natural product. The observed nanomolar cytotoxicity and microtubule destabilizing activity of the ABEF analogue provide
support for both the assigned solution conformation of (+)-spongistatin 1 and the validity of the design strategy.
’ INTRODUCTION
A and vinblastine/vincristine respectively are known to bind.6
The development of the spongistatins as possible clinical agents,
however, has not progressed given their low availability, notwith-
standing seven total syntheses of (+)-spongistatin 19 and four
total syntheses of (+)-spongistatin 2.10
Following completion of our first-generation total syntheses of
(+)-2 and (+)-1, respectively we focused on material advance-
ment to deliver more than 1 g of synthetic (+)-1, via what in
the end comprised an effective fourth-generation synthetic
route.9h,11 In addition to defining the limitations and scalability
issues along the synthetic route, a significant outcome of this
synthetic campaign was the development of a one-pot, multi-
component dithiane union tactic,12 based on the earlier efforts of
the Tietze laboratories.13 This tactic has now evolved into a new
synthetic paradigm termed Type I and Type II Anion Relay
Chemistry (ARC)14 for the rapid construction of polyketide15
and alkaloid16 natural and unnatural products.17
The spongistatins (1ꢀ9, Table 1), independently reported by
the Pettit,1 Fusetani,2 and Kitagawa3 laboratories in the early
1990s, comprise a unique family of marine macrolides that
embody remarkably complex structures possessing extraordinary
antimitotic activity. Members of this family, in particular spon-
gistatins 1 and 2 [(+)-1 and (+)-2, Table 1], rapidly attracted
broad interest in the chemical and biological communities, given
their potential as cancer chemotherapeutic agents. Spongistatin
1, recognized at the time as one of the most selective cytotoxic
agents known, has an average IC50 value of 0.12 nM against the
NCI panel of 60 human cancer cell lines.1,4 The initially observed
in vitro cell growth inhibition activity was subsequently explored
by Hamel's group for tubulin polymerization, competitive micro-
tubule assembly, and turbudity/aggregation.5 Their results re-
vealed that (+)-spongistatin 1 (1) competitively inhibits tubulin
binding of maytansine and rhizoxin, as well as GTP exchange;5,6
(2) noncompetitively inhibits tubulin binding of dolastatin 10,
halichondrin B, and vinblastine;5,6 (3) does not induce forma-
tion of GTP-independent microtubule aggregates;6 and (4)
inhibits formation of the Cys-12-Cys-201/211 cross-linking on
tubulin.7
With sufficient material for preclinical development in hand
(ca. 1 g), we turned our attention to identifying the architectural
subunits of the spongipyran skeleton responsible for the sub-
nanomolar cytotoxicity, with the goal of initiating structureꢀ
activity relationship (SAR) studies. Notwithstanding the optimized
(+)-spongistatin 1 route, construction of random analogues
On the basis of these results, Hamel and co-workers proposed
a “polyether” binding site on β-tubulin for the spongistatins
located near the vinca domain,8 distinct from but in the vicinity of
the “peptide” and “vinca” sites, where dolastatin 10/phomopsin
Received: May 23, 2011
Published: July 16, 2011
r
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Table 1. Inhibition of Cell Growth of L1210 Murine Leukemia Cells and Average GI50 Values of 60 Different Cancer Cell Linesa
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spongistatin
L1210
avg.
spongistatin
L1210
avg.
1 [(+)-1]; R = Cl, R1 = R2 = Ac
2 [(+)-2]; R = H, R1 = R2 = Ac
3 (3); R = Cl, R1 = H, R2 = Ac
4 (4); R = Cl, R1 = Ac, R2 = H
6 (5); R = R2 = H, R1 = Ac
0.03
2.0
0.13
0.85
0.83
0.10
1.10
5 (6); R = Cl, R1 = H
7 (7); R = R1 = H
8 (8); R = H, R1 = Ac
9 (9); R = Cl, R1 = Ac
0.20
2.0
0.12
1.00
0.23
0.04
1.0
3.0
0.10
0.80
0.3
a Data compiled from ref 1. GI50 values are in nM.
appeared unattractive. We were, of course, well aware of the
remarkable success of the commercial drug Halaven (eribulin),
based on the marine natural product halichondrin B, developed
by the Kishi and Eisai Inc. laboratories. We asked the question,
Is the entire structure of the large, flexible polyketide skeleton of the
spongistatins a prerequisite for biological activity?
In this, a full account, we report the design, synthesis, and
biological evaluation of two diminutive forms of (+)-spongistatin
1. Importantly, computational methods developed during this
venture led to (1) a general strategy to identify the binding
regions of flexible small-molecule macrolide natural products and,
more specifically, (2) a general design strategy that permitted the
simplification of the polyketide structure of the spongistatins
while maintaining biological activity.
For example, in Kishi's synthesis of (+)-spongistatin 1,9b as well
as in our synthesis of (+)-spongistatin 2,10b,e unexpected
epimerizations that occurred at the C(23) center of the CD
spiroketal led to congeners 10 and (ꢀ)-11 (Figure 1) that
possessed modest tumor cell growth inhibition against several
cancer cell lines [avg. GI50 of 0.2 μM for (ꢀ)-11].20c Subse-
quently, Paterson and co-workers constructed an analogue
lacking the diene side chain (12) that significantly attenuated
activity, highlighting the requirement for this structural unit.18
Later we prepared a series of glucose-based mimetics of the F
and EF rings possessing the (+)-spongistatin 2 side chain [(+)-
13 and (+)-14a].20 Although modest tumor cell growth inhibi-
tion activity was observed with (+)-13, subsequent unpublished
results suggested a different mode of action.20a,21 More re-
cently, Heathcock and co-workers reported a series of (+)-
spongistatin 2 analogues, including (+)-14b, which did not
show any activity at the tested concentrations.19 The loss of
activity led Heathcock to prepare macrolide congeners 15 and
16, employing polymethylene tethers to replace the ABCD and
CD structural segments. Observation of only modest activity
(GI50 values of 4.8 and 4.6 μM against HCT116 human colon
tumor cell lines, respectively) led Heathcock to conclude that
the CD ring may comprise a structural moiety required to
maintain the tumor cell growth inhibition.
’ SPONGISTATIN ANALOGUES: EARLY CONGENERS
Members of the spongistatin family (1ꢀ9, Table 1) possess an
array of architecturally complex structural features, including a
31-membered macrolactone ring endowed with 24 stereocen-
ters, two [6,6] spiroketals (the AB and CD rings), and one bis-
tetrahydropyranylmethane moiety (E and F rings) complete
with a diene side chain differentiated by the acetate substitution
pattern at C(5) and C(15) and, in the case of (+)-spongistatins
6ꢀ9, by an additional tetrahydropyran ring (G ring) inscribed in
the tether linking rings B and C. The cell growth inhibitory
activity against the NCI-60 DTP human tumor panel for the
natural congeners averaged at the sub-nanomolar level. Structur-
al features for optimal activity comprise the chlorine substituent
on the side chain (1 vs 2; 4 vs 6; and 6 vs 7) and an acetate
at C(5) (1 vs 3; and 6 vs 9), while structural elements not critical
for maintaining activity include the C(15) acetate and ring G
(1ꢀ5 vs 6ꢀ9). Although these observations provided useful
insights, our analogue design criteria also benefited initially from
synthetic intermediates and analogues that emanated from
the Kishi,9a,b Paterson,18 and Heathcock19 laboratories as well
as our own.20
’ ANALOGUE DESIGN: AN EF RING CONGENER POS-
SESSING A ROTATIONALLY RESTRICTED ABCD RING
SYSTEM
The lessons learned from analogues (+)-13ꢀ16, in conjunction
with the activity observed for the C(23) epi-congeners [10 and
(ꢀ)-11], led us to the hypothesis that the western perimeter of the
spongistatins constituted a significant component of the recognition
domain. We reasoned that the linker (CD) was not important and
might be replaced by a less complex unit, as long as effective
control over the conformation of the western perimeter was
maintained (i.e., the dihedral angles between rings E and F). Similar
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maximized by an increase in number of intramolecular hydrogen
bonds (Figure 2).24d In water, a “twisted” conformer is preferred
in which the oxygen atoms are oriented toward the solvent.
When DMSO and acetonitrile solvent models are employed, the
lowest energy conformer possesses a “saddle” shape. Interest-
ingly, the orientation of the EF rings in the Kitagawa solution
structure did not resemble any of these low-energy conformers.
We reasoned that the derived Kitagawa structure might well have
comprised a virtual conformation, resulting from constrained
conformational searches.
To understand further the solution behavior of (+)-spongistatin
1, molecular dynamics (MD) simulations were performed using
water as solvent, wherein changes in the dihedral angles of the
macrocycle were analyzed. The flexible regions of (+)-1 were
identified by examining (1) the dihedral angle distributions of
each Csp3ꢀCsp3 bond (Figure 3A) and (2) the long-range coupled
torsional changes during the dynamics simulation. This analysis was
performed exploiting polar coordinate graphs, in a fashion similar
to Taylor's epothilone studies,24 wherein the coupled torsions were
identified by analyzing the correlation in the torsional changes via
principal component analysis. As illustrated in Figure 3A, torsional
bonds shown in red have rigid torsions, those in green have flexible
torsions, and those in blue have torsions with intermediate
flexibility. In Figure 3B, the numbers indicate bonds where the
torsions change together, corresponding to correlated movements.
As can be seen, tethers of the CD ring are flexible, with the dihedral
angle pairs labeled with the same number changing together,
thereby permitting movement of the CD ring. Taken together,
the results of this multivariate analysis reveal (1) that the western
perimeter, including ABEF rings, has a preferred conformation
wherein each torsional angle has a preferred value and (2) that
reduction of a large, flexible molecule to a single solution structure
derived by NMR constraints in general is not feasible.
In support of these observations, MD simulations constrained
by NMR-derived distance and torsion values are known to
generate average conformations; however, the conformations
generated are often of high energy due to the constraints applied.
Equally important computational methods, relying solely on
molecular mechanics force fields, suffer from inaccurate energy
ordering of flexible organic molecules, due to the additive nature
of parametrization errors. To circumvent these issues, we devised
a computational hybrid strategy wherein the NMR-derived
interproton distances and torsional angles are used to determine
the most populated families of conformations. This effort led to
the development and implementation of a software program
termed Distribution of Solution Conformations (DISCON).
The thrust of this software package is to define a combination
of conformers that overall would provide the best match for the
time-averaged torsional angles and interproton distances obtained
by NMR. Contrary to methods developed earlier,29 DISCON
utilizes hierarchical clustering of conformations based on NMR
variables for feature selection to avoid “overfitting”,30 as well as a
genetic-algorithm-based global optimization strategy to avoid local
minima, two common problems often observed in NMR-based
structure optimization. Details and availability of this software
package for the deconvolution of NMR observables over an
ensemble of solution conformations will be provided elsewhere.31
From this analysis, we deduce that the low-energy conforma-
tions determined in water and chloroform (Figure 4aꢀd)
define the major solution conformations of (+)-spongistatin
1. The two major calculated conformations comprise twisted
Figure 1. Spongistatin analogues.
simplifying design tactics have been employed by the Kishi,22
Wender,23 Taylor,24 and Romo25 laboratories to construct
biologically active halichondrin B, bryostatin, epothilone, and
pateamine analogues, respectively.
To design a suitable tether that would orchestrate the
C(37)ꢀC(39) dihedral angles linking the spongistatin E and F
rings, we turned to molecular modeling. We were cognizant of
the solution conformation reported by Kitagawa26 during his
elegant structural assignments of 5-desacetylaltohyrtin A
(spongistatin 3, 3). To establish a baseline on the solution
conformation of (+)-spongistatin 1 [(+)-1], we carried out
repetitive Monte Carlo conformational searches on (+)-1 until
no additional families of conformers were detected. Computa-
tions utilizing the MMFF force field27 with the GB/SA solvation
model28 revealed that the energy ordering of the conformational
populations changed significantly in different solvents.
According to our calculations, in chloroform, (+)-1 adopts a
“flat” conformation in which the surface area of the molecule is
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Figure 2. Front and rear 3D views (see Supporting Information section for stereoview images) of the lowest energy conformations (IꢀIII) calculated by
conformational searches employing different solvation models and Kitagawa solution structure (IV). Blue dashed lines represent hydrogen bonds. Rings
AꢀF are shown in each conformation.
Figure 3. Flexibility of macrocyclic dihedral angles in (+)-spongistatin 1. (A) Red, rigid torsions; green, flexible torsions; and blue, torsional angles with
intermediate flexibility. (B) Numbers indicate bonds pairs where the torsions change together, corresponding to long-range movements during the
simulations.
and flat forms (57% and 13%, respectively), as illustrated in
Figure 4a,b.
The minor conformers (Figure 4c,d) are also similar to
Figure 4b, with the major difference being the orientation of the
CD ring. Pleasingly, when the four major conformations are over-
laid, a striking feature appears: the western hemispheres,
including the ABEF rings, have a common conformation
(Figure 4e). This preference can be understood by earlier
conformational studies of the bis-tetrahydropyranylmethane
system reported by Hoffmann and co-workers32 (Figure 4f),
wherein the preferred “skew” conformation minimizes the syn-
pentane interactions between the E and F ring systems. This
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Figure 4. (aꢀd) Calculated major solution conformers. (e) Overlay of these conformations, showing the conserved ABEF ring conformations, where
syn-pentane interactions (f) are important in fixing the relative conformations of E and F rings.
conformational arrangement of the E and F (+)-spongistatin 1
rings in turn facilitates an intramolecular hydrogen bond
between the E ring hydroxyl and the F ring pyran oxygen (cf.
the hydrogen bonds in Figure 2I,II).
The striking conformational similarities derived from the earlier
MD simulations and subsequent DISCON analysis, in conjunc-
tion with the biological data from the epi-C(23) spongistatin
congeners 10 and (ꢀ)-11, provided further support for the
hypothesis that the ABEF portion of the spongistatin scaffold
comprises the binding/small-molecule recognition domain that
interacts with β-tubulin.
twist of the E and F ring systems, to access the proposed
conformation of the western perimeter.
To test our conformational design strategy, we turned to
synthesis. From the synthetic perspective, 17 was easily reduced
to three fragments: phosphonium salt 18, allylstannane 19, and
aldehyde 20 (Scheme 1). The route was designed to provide
synthetic flexibility for SAR studies by a late-stage installation of
the diene side chain, exploiting intermediates in hand from our
earlier spongistatins syntheses. One of the attributes of develop-
ing preparative-scale syntheses of architecturally complex natural
products possessing significant bioregulatory properties is the
subsequent ready availability of advanced intermediates for
SAR studies. Fragments (+)-18 and aldehyde 20 were thus
envisioned to be united via a Wittig reaction. Following macro-
lactonization, side-chain attachment would be achieved via the
elegant glucalꢀepoxide union protocol developed by Evans
et al.33 during the first total synthesis in the spongistatin area.
Aldehyde 20 was particularly appealing, given Ullmann coupling
protocols.34
To explore this scenario, we first turned to analogues posses-
sing only the E and F ring systems. In silico screening of a variety
of linkers led to the biaryl ether tether in 17 (Scheme 1, red),
which would be suitable to orchestrate the relative conformational
Scheme 1. Retrosynthetic Analysis of EF Ring Analogue 17
We began the synthesis of 17 with (+)-21, from our second-
generation (+)-spongistatin 1 synthesis.9f We envisioned that
(+)-21 could be readily converted to the corresponding F-ring
dihydropyran (+)-22 by treatment with trifluoromethane-
sulfonic anhydride (Tf2O), followed by elimination of TfOH
(Table 2). In the event, use of lithium diisopropylamide (LDA)
provided an inseparable mixture (ca. 1:1) of (+)-22 and a
compound whose structure was tentatively assigned as 23.
Reasoning that a bulkier base might favor the desired compound,
use of KHMDS resulted in a 3:1 mixture of (+)-22 and 23, albeit
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Table 2. Selectivity in the Preparation of F Ring Pyran
Scheme 3. Preparation of Biaryl Ether 20
entry
base
LDA
temp (ꢀC)
yield (%)
ratio (+)-22:23
1
2
3
ꢀ45 to ꢀ20
ꢀ45 to ꢀ20
ꢀ78 to ꢀ30
56
35
72
1:1
3:1
by Ullmann coupling34 with 4-hydroxybenzaldehyde to yield 20.
While the efficiency of the last step was only modest, we moved
forward to explore the viability of this tether.
KHMDS
NaHMDS
12:1
Wittig union36 of (+)-18 with 20 provided the Z-olefin
geometry, albeit in low yield (Scheme 4). With studies to
improve both the Ullmann and Wittig unions underway, we
in modest yield (35%). Cooling the reaction mixture to ꢀ78 ꢀC
led to exclusive formation of (+)-22, although the reaction did
not proceed to completion. In an effort to increase the efficiency,
we examined the use of the less basic NaHMDS. Pleasingly,
significant improvement in selectivity (12:1) was observed
to provide (+)-22 in 72% overall yield. With reliable access
to glucal (+)-22, removal of the benzyl protecting groups
employing lithium 4,40-di-tert-butylbiphenylide (LiDBB) pro-
ceeded smoothly to provide diol (+)-24 in good yield
(Scheme 2). Selective conversion of the primary hydroxyl to
furnish the 2,4,6-triisopropylbenzenesulfonate (trisylate) (+)-25
initially proved problematic when the bulky base 2,6-di-tert-
butyl-4-methylpyridine (DTBMP) was employed. The low
reactivity of (+)-24 led us to screen a series of the bases. Triethyl-
amine with DMAP at 0 ꢀC proved optimal, selectively furnishing
(+)-25 in 83% isolated yield, which in turn was treated with
LiI in the presence of 2,6-lutidine to generate iodide (+)-26. The
secondary hydroxyl was then converted to the TES ether, followed by
displacement of the iodide in (+)-27 with triphenylphosphine to
furnish Wittig partner (+)-18 in nearly quantitative yield.
Scheme 4. Attempted Removal of tert-Butyl Group in (+)-30
The requisite biaryl ether tether 20 (Scheme 3) was con-
structed via condensation of 4-bromobenzoic acid with tert-
butanol, mediated by 1,10-carbonyldiimidazole (CDI),35 followed
Scheme 2. Preparation of (+)-18
proceeded with removal of the tert-butyl ester. Unfortunately,
various conditions, including treatment of (+)-30 with
TMSOTf, resulted only in decomposition, presumably due to
the incompatibility of the enol ether with the required mild
Lewis acidic conditions.
Concurrent with studies to access 17 from (+)-13, we also had
access to EF Wittig salt (+)-31 (Scheme 5), again developed in
Scheme 5. Second-Generation Retrosynthesis of EF Ring
Analogue 17
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conjunction with our gram-scale synthesis of (+)-spongistatin 1.10e
A second-generation approach to the spongistatin EF analogue 17
was thus initiated on the basis of the availability of (+)-31. In the
forward sense, we envisioned a biaryl linker now possessing a
triisopropylsilyl (TIPS) group to protect the carboxylic acid, in view
of our experience with the tert-butyl ester. Construction of this
biaryl ether tether, employing the Evans-modified Ullmann aryl
ether synthesis,37 proved to be compatible with the labile TIPS
ester (Scheme 6).
Scheme 8. Macrolactonization of 35
Scheme 6. Preparation of 32
Union of 32 with EF Wittig salt (+)-31 was achieved upon
deprotonation with MeLi LiBr (Scheme 7). Addition of 32 pro-
3
vided 34 as in an inseparable mixture (4:1) of Z- and E-olefin
isomers. The lack of complete Z-selectivity was surprising, given
that the identical conditions in the gram-scale synthesis of (+)-
spongistatin 1 led exclusively to the Z-olefin. The mixture of
olefin isomers, without separation, was then subjected to TBAF
(3 equiv) to effect deprotection of both the TIPS ester and the
F-ring TES ethers; seco-acid 35, as a mixture of olefin Z:E isomers
(4:1), was obtained in 67% yield.
analysis. Presumably, following macrocyclization, the molecule
adopts a conformation which facilitates acylation at C(42).
Reduction of the amount of each reagent [15 equiv of i-Pr2NEt,
9 equiv of 2,4,6-TBCCl, and 20 equiv of DMAP, Scheme 8B]
furnished the desired macrocycle (+)-37, along with a minor
inseparable impurity.
Scheme 7. Preparation of Macrolactonization Precursor 35
Global deprotection next yielded a mixture of products (11:1).
Unfortunately, the major product was not the desired 17-Lactol
(Scheme 9) but instead was identified as the E-ring-opened
17-Ketone, assigned on the basis of a 13C NMR carbonyl resonance
at 211.4 ppm. For comparison, the hemiketal carbon resonance
of (+)-37 appears at 100.5 ppm.
Isodesmic calculations,38 employing 17-Ketone, (+)-1, and
the corresponding C(1) seco-acids, were performed in a vacuum
Scheme 9. Global Deprotection of (+)-37 under Conditions
Followed in the Gram-Scale Spongistatin 1 Synthesis
The macrocyclization conditions that had proven successful
in the preparation of (+)-spongistatin 1 [60 equiv of i-Pr2NEt,
20 equiv of 2,4,6-trichlorobenzoyl chloride (TBCCl) in toluene
at room temperature, then DMAP, 90 ꢀC, Scheme 8A] were
explored first. A single product containing a Z-olefin was
generated, presumably due to the strain associated with incor-
porating an E-olefin into the macrocycle. The desired macrocycle
(+)-37, however, was not the product, but instead trichloro-
benzoate 36, tentatively assigned on the basis of NMR and MS
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with the MMFF force field. The results revealed the increase in
strain energy upon macrocyclizations of (+)-1 and 17-Lactol to
be 5.8 and 45.2 kJ/mol, respectively. In a similar fashion,
calculations employing the 17-Lactol and 17-Ketone revealed
the release in strain energy upon opening of the pyran ring of
17-Ketone to be 11.2 kJ/mol. Although not definitive, these
calculations suggest that, upon acidic global deprotection, the
macrocyclic strain energy leads to opening of the E ring. We
therefore decided to reinvestigate our design strategy.
Scheme 10. Retrosynthesis of ABEF Ring Analogue
’ ADVANCED ANALOGUE DESIGN: LESSONS
LEARNED LEAD TO A (+)-SPONGISTATIN CONGENER
POSSESSING NANOMOLAR TUMOR CELL GROWTH
INHIBITORY ACTIVITY
The design and synthesis of biaryl ether EF analogue 17-lactol
led to an important lesson: consideration of ring strain energy is
critical in the design of macrocyclic analogues. Although a linker
may fix the overall conformation, the induced ring strain
associated with the designed macrocycle may not be compatible.
To rectify this issue, we turned to more flexible polymethylene
tethers (Scheme 10). We also chose to incorporate an internal
hydrogen bond acceptor in the form of a lactone carbonyl that
might participate in a H-bond to the C(42)-hydroxyl on the F
ring, in an effort to orchestrate the conformation of the (+)-
spongistatin western binding/recognition domain. Our goal was
to lower the overall flexibility observed in both our solution
conformational studies of (+)-1 and the Heathcock cyclic
analogues (15 and 16, Figure 1). Finally, we chose to include
the AB spiro-ring moiety to expand the structural elements in an
attempt to maximize tubulin binding.
Iterative in silico conformational searches, employing a series
of tethers of different lengths with hydrogen bond acceptors, led
to structure 38 (Scheme 10, red), possessing a tether comprising
a Z-olefin, in conjunction with a lactone moiety and a series of
methylene units to mimic the span between ring B and the
replaced CD spiroketal system. Importantly, the strain energy
associatedwiththedesigned29-membereddiolide38 (9.1kJ/mol)
was found to be nearly the same as the ring strain energy of
(+)-spongistatin 1 (8.3 kJ/mol).
available from our gram-scale synthesis of (+)-spongistatin 1.
That is, Wittig union involving EF phosphonium salt (+)-31 and
now aldehyde 47, followed by macrolactonization and global
deprotection, would lead to 38 (Scheme 10). Tether 47, in turn,
would be constructed from three fragments: known AB ring
aldehyde 40,39 Wittig salt 41, and primary acid 44,40 the latter
derived from δ-valerolactone.
Toward this end, Wittig union of (ꢀ)-40 with 41 led to (ꢀ)-
42 in 51% yield, along with the deacetylated congener (ꢀ)-42a,
which was readily reacetylated to furnish (ꢀ)-42 (Scheme 11);
the overall yield was 63%. Given our earlier experience with
(+)-30 (Scheme 4), the tert-butyl ester was converted to the
TIPS ester by a three-step protocol (ca. 98% yield for the three
steps). Known acid 44,38 the remaining component, was then
appended to (ꢀ)-43 via esterification. Removal of the PMB
group followed by oxidation41 delivered the desired tether
(ꢀ)-47 in good yield.
From the synthetic perspective, analogue 38 was envisioned to
arise from advanced AB and EF ring intermediates, again
Scheme 11. Synthesis of (ꢀ)-47
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Scheme 12. Preparation ABEF Ring Analogue (ꢀ)-38
Figure 5. (aꢀc) Calculated major solution conformers of ABEF
analogue (ꢀ)-38. (d) Overlay of the major solution conformation a
with (+)-spongistatin 1 conformer seen in Figure 4b.
incorporates three additional H-bonds, in this case with the F
ring hydroxyl selecting the C(48) hydroxyl [(+)-spongistatin 1
numbering] group on the side chain, rather than with the lactone
carbonyl on the linker. Finally, the minor conformer family found
(Figure 5c, 8%) incorporates a H-bond between the linker
carbonyl and the C(48) [(+)-spongistatin 1 numbering] side-
chain hydroxyl. Overall, the conformations are quite rigid at the
ABEF ring juncture, giving the side chain a more preferential
alignment compared to the (+)-1 structures. Overlay of the three
conformations of (ꢀ)-38 (Figure 5aꢀc) with the identified
solution conformations of (+)-1 (Figure 4aꢀd) reveals that they
all possess a similar western conformation (Figure 5d).
’ BIOLOGICAL STUDIES
Not surprisingly, the first-generation EF ring analogue, isolated as
a 11:1 mixture of structural isomers (17-Ketone and 17-Lactol)
favoring the 17-Ketone, revealed low biological activity in the
cell-based growth inhibition assay (Table 1). Pleasingly, however,
the ABEF ring analogue (ꢀ)-38, demonstrated by NMR to
comprise a good conformational mimic of the western perimeter
of (+)-1, displayed nanomolar inhibitory activity (Table 3).43 The
question that now arose, given that a substantial proportion of the
natural product had been deleted, became, Does this analogue have
the same mechanism of action as (+)-spongistatin 1?
Table 3. Cell Growth Inhibition (IC50, nM) with Spongista-
tin 1 and the Synthesized Analogues
Union of (ꢀ)-47 with EF Wittig salt (+)-31 proceeded
smoothly to furnish (ꢀ)-48 (Scheme 12) in 64% yield, in
this case exclusively as the Z-olefin. Removal of the TES and
TIPS groups employing TBAF in THF next provided seco-acid
(ꢀ)-49, which upon Yamaguchi lactonization42 followed
by global deprotection employing HF in CH3CN—conditions
identical to those employed in our large-scale (+)-spongistatin 1
synthesis10e—completed the construction of (ꢀ)-38, the ABEF
analogue. The yield for the final two steps was 48%.
The solution conformation of (ꢀ)-38 was investigated by
DISCON computational and NMR studies, similar in detail to
those we employed to define the solution conformations of (+)-
spongistatin 1. Three solution conformational families were
found in CHCl3 (Figure 5aꢀc), wherein the western ABEF
perimeters of all conformers effectively recapitulate the western
ABEF perimeter conformation observed in (+)-1. The major
solution conformer (Figure 5a, 59%) incorporates two hydrogen
bonds in addition to the H-bonds within the EF system. In
accord with our design, the F ring hydroxyl makes a H-bond with
the newly introduced lactone carbonyl, as well as the C(10) B
ring hydroxyl, further fixing the conformation of the AB ring. The
second major conformational family (Figure 5b, 33%)
MDA-MB-435
HT-29
H522-T1
U937
(+)-1
(ꢀ)-38
17
0.0225
82.8
0.058
161.2
>1000
0.16
0.059
60.5
297.2
>1000
>1000
>1000
To answer this question, the effects of (+)-1 and ABEF
analogue (ꢀ)-38 on cell cycle distribution were evaluated in a
standard cell cycle analysis employing U937 lymphoma cells. As
shown in Figure 6A, both (+)-spongistatin 1 (1) and analogue
(ꢀ)-38 led to significant enrichment of cells in the G2/M phase
of the cell cycle (G2/M peaks are indicated by arrows), con-
sistent with the known mechanism of action of (+)-spongistatin
1 to target microtubule architecture. To ascertain further that
analogue (ꢀ)-38 retains the same mechanism of action as (+)-1,
both compounds were evaluated in an in vitro tubulin polymer-
ization assay. As shown in Figure 6B, analogue (ꢀ)-38 inhibits
tubulin polymerization in a dose-dependent manner, similar to
(+)-1. The higher concentration (i.e., 30 μM) required for
analogue (ꢀ)-38 to inhibit tubulin polymerization reflects the
lower potency of the analogue as compared to the extraordinarily
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Figure 6. (A) G2/M cell cycle effects of (+)-spongistatin 1 and (ꢀ)-38. U937 cells were treated with either (+)-1 or the (ꢀ)-38 analogue for 18 h.
Samples were subjected to flow cytometric cell cycle analysis. Data shown represent relative number of cells (Y axis) as a function of fluorescence
intensity representing DNA content (X axis). (B) Inhibition of in vitro tubulin polymerization. An in vitro tubulin polymerization assay was carried out in
the presence of indicated compounds and concentrations. Tubulin polymerization curves shown here represent the relative amount of tubulin polymer
(OD340, Y axis) over time (X axis).
potent (+)-1. Vinblastine, a microtubule destabilizing agent, and
paclitaxel, a tubulin stabilizing agent, were included as controls.
Thus, like (+)-1, the designed analogue (ꢀ)-38 possesses
significant microtubule destabilizing activity.
Present Addresses
‡Bristol-Myers Squibb, Inc.
#Chemistry Department, Tufts University
’ ACKNOWLEDGMENT
’ SUMMARY
Financial support was provided by the NIH (NCI)
through Grant No. CA-70329. C.A.R. was supported by an
American Cancer Society-Michael Schmidt Postdoctoral
Fellowship.
The feasibility of designing a diminutive congener of
(+)-spongistatin 1, (ꢀ)-38, wherein approximately one-third
of the parent compound is replaced by a simplifying tether that
retains significant microtubule destabilizing activity (ca. 60 nM
for U937 cell lines), has been demonstrated. To achieve this goal,
we introduced a new computational method (DISCON) that led
to a design strategy based on the hypothesis that the pharmaco-
phoric elements of biologically active natural products are likely
to reside on conformationally rigid portions of the molecule.
Significantly, this synthetic effort led to the elimination of some
30 steps compared to the synthesis of (+)-spongistatin 1.11 Finally, a
general strategy for the design of simplified bioactive analogues of
large, flexible natural products has been developed and validated.
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S
Supporting Information. Spectroscopic and analytical
b
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smithab@sas.upenn.edu
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