Latrunculin analogues with improved biological profiles by "diverted total synthesis": Preparation, evaluation, and computational analysis

Article abstract of DOI:10.1002/chem.200601136

Deliberate digression from the blueprint of the total syntheses of latrunculin A (1) and latrunculin B (2) reported in the accompanying paper allowed for the preparation of a focused library of "latrunculin-like" compounds, in which all characteristic str

Full text of DOI:10.1002/chem.200601136

FULL PAPER
DOI: 10.1002/chem.200601136
Latrunculin Analogues with Improved Biological Profiles by “Diverted Total
Synthesis”: Preparation, Evaluation, and Computational Analysis
Alois Fürstner,*[a] Douglas Kirk,[a] Michal D. B. Fenster,[a] Christophe Aïssa,[a]
Dominic De Souza,[a] Cristina Nevado,[a] Tell Tuttle,[a] Walter Thiel,[a] and
Oliver Müller[b]
Abstract: Deliberate digression from
the blueprint of the total syntheses of
latrunculin A (1) and latrunculin B (2)
reported in the accompanying paper al-
lowed for the preparation of a focused
library of “latrunculin-like” com-
pounds, in which all characteristic
structural elements of these macrolides
were subject to pertinent molecular
much more widely practiced chemical
modification of a given lead compound
by conventional functional group inter-
conversion. A computational study was
carried out to rationalize the observed
effects. The analysis of the structure of
the binding site occupied by the indi-
vidual ligands on the G-actin host
shows that latrunculin A and 44 both
have similar hydrogen-bond network
strengths and present similar ligand
distortion. In contrast, the H-bond net-
work is weaker for latrunculin B and
the distortion of the ligand from its op-
timum geometry is larger. From this,
one may expect that the binding ability
follows the order 1 ꢀ 44 > 2, which is
in accord with the experimental data.
Furthermore, the biological results pro-
vide detailed insights into structure/ac-
tivity relationships characteristic for
the latrunculin family. Thus, it is dem-
onstrated that the highly conserved
thiazolidinone ring of the natural prod-
ucts can be replaced by an oxazolidin-
edit
A
ACHTREoUGN ne moiety, and that inversion of the
ed derivatives of 1 and 2 were essen-
tially devoid of any actin-binding ca-
pacity, the synthetic compounds pre-
sented herein remain fully functional.
One of the designer molecules with a
relaxed macrocyclic backbone, that is
compound 44, even surpasses latruncu-
lin B in its effect on actin while being
much easier to prepare. This favorable
result highlights the power of “diverted
total synthesis” as compared to the
configuration at C16 (latrunculin B
numbering) is also well accommodated.
From a purely chemical perspective,
this study attests to the maturity of
ring-closing
alkyne
metathesis
(RCAM) catalyzed by a molybdenum
alkylidyne complex generated in situ,
which constitutes a valuable tool for
advanced organic synthesis and natural
product chemistry.
Keywords: actin · chemical biology ·
computational chemistry · macro-
lides · metathesis · total synthesis
Introduction
rapid disassembly of the actin cytoskeleton.[1,2] In terms of
specificity and efficacy, this effect is reminiscent of genetic
knockout experiments that inactivate a single cellular con-
stituent. As a result, the latrunculins enjoy widespread use
as probe molecules in chemical biology;[3] however, their
mode of action is by no means fully understood, not least
because insights into structure/activity relationships (SAR)
are missing. This lack of information is due, in part, to the
fact that essentially all post facto chemical modifications of
1 and 2 engendered a complete loss of their physiological
activity.[4]
Actin, as the most abundant protein, determines the
shape and mechanical properties of eukaryotic cells, effects
cell locomotion and strength development in muscles, and is
involved in motility processes as fundamental as cytokinesis,
exocytosis and endocytosis.[5] The latrunculins are known to
Incubation of eukaryotic cells with low micromolar concen-
trations of the marine natural product latrunculin A (1) or
its ring-contracted congener latrunculin B (2) results in the
[a] Prof. A. Fürstner, Dr. D. Kirk, Dr. M. D. B. Fenster, Dr. C. Aïssa,
Dr. D. De Souza, Dr. C. Nevado, Dr. T. Tuttle, Prof. W. Thiel
Max-Planck-Institut für Kohlenforschung
45470 Mülheim/Ruhr (Germany)
Fax : (+49)208-306-2994
E-mail: fuerstner@mpi-muelheim.mpg.de
[b] Univ.-Doz. O. Müller
Max-Planck-Institut für Molekulare Physiologie
44227 Dortmund (Germany)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2007, 13, 135 – 149
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135

structural motif in nature but is so prominently featured
throughout the entire latrunculin family.[2]
Furthermore, the crystal structure shows the fit between
the macrocyclic loop of 1 and the hydrophobic region of the
binding site, even though the hydrocarbon chain from C5 to
C7 forms only few contacts and is partly exposed to sol-
vent.[6] This may explain why nature has evolved the ring
contracted congener latrunculin B (2) missing one of the
double bonds alongside with latrunculin A (1) as a similarly
potent actin binder.
form 1:1 complexes with the actin monomers (globular
actin, G-actin) that are incapable of polymerizing to the
intact protein filament network (fibrous actin, F-actin).
Their binding site is located near the nucleotide binding
cleft between the four subdomains of the protein and was
structurally characterized at 2Š resolution by crystal struc-
ture analysis of latrunculin A (1) bound to its protein host
(Figure 1).[6,7] This investigation indicates that binding of 1
exploits the very mobility of the protein loops of G-actin,
thereby influencing critical subdomain contacts at a minimal
penalty to its own ligation near the better ordered base of
the cleft.
A more detailed analysis of the interactions between 1
and G-actin reveals several noteworthy features: While the
2-thiazolidinone group fits neatly into the binding pocket, it
does not seem to engage its conspicuous sulfur atom in the
hydrogen bond network formed by the other heteroele-
ments.[8] Specifically, the S atom does not entertain any im-
mediate interactions below 4 Š except for an intramolecular
bond to the C17 hydroxy group (3.478 Š) of its own back-
bone. The two other closest residues are a water molecule
(4.084 Š) and R206 of the protein (4.054 Š). This abstinence
of the sulfur atom is highly surprising if one considers that
the 2-thiazolidinone ring constitutes an otherwise unknown
The published crystal structure[6] can also be interrogated
in stereochemical terms. Thus, the configuration of C16 de-
termines the orientation of the thiazolidinone ring relative
to its hydrogen bonding partners and may hence affect the
binding constant. Under this premise it is surprising that a
recent collection of the producing sponge Negombata mag-
nifica has led to the isolation of small amounts of 16-epi-la-
trunculin B (3), which differs from all other members of this
family in the absolute stereochemistry at this particular
site.[9] Unfortunately, however, the actin-binding capacity of
3 was not reported, even though it is known that this com-
pound retains significant cytotoxicity and exhibits antiviral
properties as well.
The information gathered from the crystal structure
analyACHTREsUGN is of 1 bound to G-actin is graphically summarized in
Figure 2. From this, it seems highly likely that modifications
of the chemically accessible heteroelements will disrupt the
tight hydrogen bonding pattern, which may explain why pre-
vious attempts to improve actin binding by derivatization of
1 or 2 essentially met with failure.[4a] Rather, we presumed
that meaningful SAR-studies should focus on the elucida-
tion of the role of the sulfur atom, the macrocyclic frame,
and the chiral center C16. Any such structural editing, how-
ever, is difficult to achieve if one starts from the natural
products themselves; de novo
synthesis seems to be much
more appropriate to garner
releACHTERvUNG ant insights. Because our
total syntheses of 1–3 reported
in the accompanying paper are
flexible by design,[11] we felt in
a strong position to venture
into such an endeavor at the
chemistry/biology
interface.
The results of our “diverted
total synthesis”[12] campaign
are summarized below.[13]
Results and Discussion
Preparation of a focussed li-
brary: Our total syntheses of
latrunculin A (1), its ring con-
tracted congener latrunculin B
(2) and the recently discovered
16-epi-analogue 3 hinge upon
Figure 1. Pertinent part of the crystal structure of the G-actin/latrunculin A (1) complex, together with the pu-
tative hydrogen-bonding network responsible for ligand binding according to ref. [6].
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Chem. Eur. J. 2007, 13, 135 – 149

Latrunculin Analogues
FULL PAPER
clic ketones were chosen to address the specific role, if any,
of the sulfur atom and should also reveal whether the con-
figuration of C16 correlates with biological activity. The dif-
ferent acid parts allow one to fine-tune the lipophilicity of
the compounds and to modulate the rigidity of the macrocy-
clic perimeters. Their syntheses (Schemes 1 and 2) followed
Figure 2. Graphical summary of the putative bonding situation between
latrunculin A (1) and G-actin as evident from the crystal structure analy-
sis reported in ref. [6], together with the published hydrogen bond
lengths.[10]
the convergent assembly of these targets from three building
blocks and are therefore inherently modular.[11–14] Since the
individual components are readily amenable to structural
modification, the underlying synthesis blueprint might allow
for the preparation of a focused library of analogues ade-
quate for investigations of the structural requirements for
actin binding.
Scheme 1. a) Triphosgene, Et3N, CH2Cl2, 08C, 99%; b) PMBBr, NaH,
THF, ꢁ158C, 56%; c) i) KOH, 1,4-dioxane/H2O, 97%; ii) (MeO)-
MeNH·HCl,
(benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium
hexafluorophosphate (BOP), Et3N, CH2Cl2, 99%; d) MeMgBr, THF,
ꢁ408C, 96% (ee 77%, crude), then recrystallization form tert-butyl
methyl ether, ee ꢀ 97%.
To this end, a matrix of four heterocyclic building blocks
4–7, five acid segments 8–12, and two different aldehyde
components 13 and 14 was prepared that allow for the as-
sembly of a sizeable number of “latrunculin-like” macro-
lides via the established routes (Figure 3).[13,14] The heterocy-
established routes, with the use of cheap, non-toxic and
benign [FeCAHTRE(UNG acac)3] as catalyst in the cross coupling step lead-
ing to 9 being particularly noteworthy.[15]
With regard to the aldehyde segment, the methyl branch
at C8 (Lat-B numbering) was
removed in synthon 14 relative
to the parent compound 13.
This seemingly minor modifi-
cation pays off in preparative
terms since access to 14 re-
quired only three high yielding
steps starting from 5-heptynal
29 and is easily scaleable
(Scheme 3).
The assembly process fol-
lowed the protocols developed
in the total synthesis of 1–3 as
described in detail in the ac-
companying paper,[11] although
not all steps were fully opti-
mized for each individual ana-
logue prepared during this
campaign.
A
representative
case is outlined in Scheme 4.
Thus, the titanium aldol reac-
tion[16] of the serine derived
ketone 7 with the “nor-methyl”
aldehyde 14 gave alcohols 32
and 33 in a 1.1:1 ratio which
were separable by flash chro-
matography. The stereochemi-
Figure 3. Matrix of building blocks used for the preparation of the latrunculins and analogues. Structural modi-
fications relative to the parent natural products are color-coded in red.
Chem. Eur. J. 2007, 13, 135 – 149
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137

A. Fürstner et al.
Scheme 2. a) LiCl, HOAc, MeCN, reflux, 60%; b) [FeCATHRE(UNG acac)3] (10 mol%),
THF, ꢁ308C, 78%; c) NaOH, MeOH, 74%.
Figure 4. Molecular crystal structure of compound 33 in the solid state.
Anisotropic displacement parameters are drawn at the 50% probability
level.
Scheme 3. a) (ꢁ)-Ipc2B(allyl), Et2O, ꢁ1008C; b) TBSCl, imidazole, DMF,
C
(Z)-configured a,b-unsaturated carboxylic acid 9 with for-
mation of the thermodynamically more stable (E)-config-
ured ester 37,[18] the secondary
hydroxy group of 35 was in-
verted by means of a Mitsuno-
bu reaction/saponification se-
quence,[19] thereby ensuring
convergence of the assembly
line.
Cyclization of 36 by ring-
closing
alkyne
metathesis
proceeded
(RCAM)[20,21]
smoothly, affording cyclo-
alkyne 38 in 71% yield, pro-
vided that the molybdenum
complex [Mo{N(tBu)(Ar)}3]
AHCTREUNG
AHCTREUNG
(40, Ar=3,5-dimethylphenyl)
activated in situ with CH2Cl2,
as previously described by our
group,[22] was used as the cata-
lyst (Scheme 5); in striking
contrast, attempted cyclization
with the aid of the Schrock al-
Scheme 4. a) TiCl4, (iPr)2NEt, CH2Cl2, ꢁ788C, 58% (d.r. 1.1:1); b) i) aq. HCl (1m), THF; ii) MeOH, CSA cat.,
34 (92%), 35 (88%); c) i) diisopropyl azodicarboxylate (DIAD), p-nitrobenzoic acid, THF; ii) K2CO3, MeOH/
H2O, 49% (over both steps); d) i) Tf2O, pyridine, CH2Cl2, ꢁ78 ! ꢁ458C; ii) sodium salt of acid 9, [15]crown-
5, THF, 08C ! RT, 70%; e) 2,4,6-trichlorobenzoic acid chloride, Et3N, acid 9, DMAP cat., toluene, 71%.
ꢂ
kylidyne [(tBuO)3W CCMe3]
(41)[23] met with failure, thus
attesting to the superior func-
cal assignment was unambiguous on the basis of a crystal
structure analysis of the minor product (Figure 4).
tional group compatibility of the molybdenum-based
system. Final Lindlar hydrogenation of cycloalkyne 38 fol-
lowed by global deprotection of the resulting alkene with
cerium ammonium nitrate (CAN) furnished product 39 in
good overall yield. This particular compound differs from la-
trunculin B (2) in three structural aspects: it contains a pe-
ripheral oxazolidin-2-one rather than a thiazolidin-2-one
ring, shows the opposite configuration at the attachment
point C16, and lacks the two methyl branches on the macro-
cyclic loop. A number of related, fully synthetic “latruncu-
lin-like” products were prepared analogously (Figure 5) with
The two stereomers were processed separately by acid
catalyzed cleavage of the TBS groups, leading to the sponta-
neous cyclization of the corresponding tetrahydropyran
rings which were locked as methyl glycosides. Compound 34
was then converted into diyne 36 as the substrate for the en-
visaged ring-closing alkyne metathesis (RCAM) via the cor-
responding triflate.[11,14] Since attempted conversion of the
diastereomeric alcohol 35 into diyne 36 under Yamaguchi
conditions[17] led to the concomitant isomerization of the
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Latrunculin Analogues
FULL PAPER
Scheme 6. a) CAN, MeCN/H2O, 80%.
Scheme 5. a) Complex 40 (25 mol%), toluene/CH2Cl2, 808C, 71%; b) H2
(1 atm), Lindlar catalyst, CH2Cl2, quant.; c) CAN, MeCN/H2O, 93%.
the structural changes relative to the parent latrunculins
being highlighted in red for easier survey.
Additional compounds for testing were obtained by “pre-
mature escape” from the assembly line. Specifically, deriva-
tive 48 representing the “alkynylogous” congener of latrun-
culin B (2) was accessible by deprotection of cycloalkyne 53
as the key intermediate of our total synthesis of
2
(Scheme 6).[11,14] Due to its very strained bicyclic edifice,
however, this compound had only a limited lifetime. To ad-
dress the question whether a macrocyclic backbone is neces-
sary at all for actin binding, analogues 50 and 51 were pre-
pared from building blocks 18 and 17,[13] respectively, by the
standard esterification/deprotection manifold (Scheme 7).
Finally, the bare macrocycle 52 devoid of the entire hetero-
cyclic domain present in latrunculin B was prepared as a
“negative control” for the bioassay on treatment of diyne 56
with the molybdenum catalyst 40 in toluene/CH2Cl2 under
Scheme 7. a) Acid 9, diethyl azodicarboxylate (DEAD), PPh3, benzene,
61%; b) CAN, MeCN/H2O 2:1, 49%; c) heptanoic acid, DEAD, PPh3,
benzene; d) CAN, MeCN/H2O 2:1.
the usual conditions, followed by Lindlar hydrogenation of
the resulting cycloalkyne 57 (Scheme 8).
Evaluation of the actin-binding
properties: It is well establish-
ed in the literature that incu-
bation of non-muscular cells
with 1 or 2 leads to almost in-
stantaneous
morphological
changes and shape malforma-
tions, which constitute a con-
venient
phenomenological
read-out for actin binding.[1,3]
This type of assay was used for
the evaluation of the com-
pounds prepared above using
the NIH3T3 fibroblast cell-
line. The actin cytoskeleton of
these cells was visualized by
staining with fluorescence-
marked phalloidin, whereas
their nuclei were stained in
blue with 2-(4-amidinophenyl)-
Figure 5. Collection of fully synthetic latrunculin analogues used for the actin-binding assays.
Chem. Eur. J. 2007, 13, 135 – 149
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139

A. Fürstner et al.
Table 1. Microfilament disrupting activity of Lat-A (1), Lat-B (2), 16-epi-
Lat-B (3), and their fully synthetic analogues.[a]
Compound
Effective concentration
1 mm
5 mm
10 mm
1
+
+
ꢃ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
++
ꢃ+
+
+++
ꢃ++
++
++
ꢃ+
+
44
42
2
39
3
43
45
46
47
48
50
51
49
52
+
Scheme 8. a) Complex 40 (10 mol%), toluene/CH2Cl2, 808C, 70%; b) H2
(1 atm), Lindlar catalyst, CH2Cl2, 70%.
+
ꢃ
ꢃ
+
ꢃ
+
6-indolecarbamidine hydrochloride (DAPI) (Figure 6, mi-
crograph I).
ꢃ
+
ꢃ
+
ꢁ
+
ꢁ
ꢃ
ꢁ
ꢃ
ꢁ
ꢁ
ꢁ
ꢁ
[a] Abbreviations: ꢁ, no effect; ꢃ, weak effect: unaltered cell number,
intact cell morphology, filamentous and slightly disrupted actin; +, sig-
nificant effect: less than 80% viable cells, spindle-shaped cell morpholo-
gy, filamentous and disrupted actin ; ++, strong effect: less than 50%
viable cells, poly-nuclear cells, more disrupted than filamentous actin;
+++, very strong effect: less than 20% viable cells, poly-nuclear,
rounding and isolated cells, disrupted actin (compare representative mi-
crographs in Figure 6).
As can be seen from this compilation, all macrocyclic
compounds, except the bare lactone 52 devoid of the hetero-
cyclic domain, were found to induce depolymerization of F-
actin at 10 mm concentration. Thus, the implemented struc-
tural modifications are well accommodated and the non-nat-
ural analogues remain functional. This result is highly signif-
icant if one recalls that virtually all post-facto chemical
modifications of the latrunculins previously reported in the
literature resulted in a complete loss of actin depolymeriza-
tion capacity.[4]
Figure 6. Fluorescence micrographs (250”) of NIH3T3 fibroblast cells
show that compound 44 exerts a stronger effect than the natural product
Lat-B (2). The actin filament is stained with fluorescence-marked phalloi-
din, the nuclei with DAPI. I: untreated; II: after incubation with 2
(5 mm); III: after incubation with 44 (5 mm); IV: after incubation with 44
(10 mm).
A closer look at the data set reveals further salient fea-
tures. Most strikingly, removal of the seemingly unfunctional
methyl branches at C3 and/or C8 of the macrocyclic ring of
2 leads to a significant increase in potency. As evident from
the micrographs shown in Figure 6, the bis-nor-Lat-B deriva-
tive 44 clearly surpasses the parent compound latrunculin B
(2) in its effect on actin, reaching a potency previously
known only for latrunculin A as the most active natural
product of this series (cf. Table 1). This noteworthy result
has important implications: Since 44 is much easier to pre-
pare than both 1 and 2 by a route that has only 11 steps in
the longest linear sequence,[13] this particular analogue may
become available through total synthesis in sufficient quanti-
ty at a competitive price and might therefore help to ensure
a constant supply of a fully functional and highly potent
actin-binding probe molecule. Access to the natural latrun-
culins by harvesting and extracting the producing sponge is
limited and gets increasingly difficult because Negombata
magnifica populates the highly sensitive and protected eco-
system of the coral reefs of the Red Sea.[24]
In accordance with literature data,[1,3] latrunculin A (1)
turned out to be more effective than its ring contracted con-
gener latrunculin B (2). After this calibration of the assay,
all novel analogues were screened at different concentra-
tions. Representative micrographs are depicted in Figures 6
and 7, and the resulting semi-quantitative data are summar-
ized in Table 1.
Figure 7. Fluorescence micrographs (250”) showing the effect of synthet-
ic latrunculin derivatives on NIH3T3 fibroblasts: I: after incubation with
16-epi-LAT B (3) (5 mm); II: after incubation with 39 (5 mm).
The other structural modifications engender a slight de-
crease in activity. Specifically, all compounds belonging to
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Latrunculin Analogues
FULL PAPER
the 16-epi-series are somewhat less active than their parent
anaACHTREUNGlogues. This includes naturally occurring 16-epi-latruncu-
and the complex with the best binding energy from each
docking simulation was pre-minimized in CHARMM.[25–27]
The structures resulting from the force field minimization
were optimized at the hybrid quantum mechanical/molecu-
lar mechanical (QM/MM) level of theory with the modular
program package chemshell.[28,29] The QM energy and gra-
dients were provided by MNDO99,[30] with MNDO/H[31,32]
as the QM level of theory. CHEMSHELLꢁs internal force
field driver supplied the MM energy and gradients, using
the CHARMM parameter and topology data.[33–35] No elec-
trostatic cut-offs were employed in the QM/MM calcula-
tions. Electrostatic embedding[36] with the charge-shift
scheme[29,37] was used to couple the QM and MM regions.
Full details of the computational methodology employed in
this study are available in the Supporting Information.
In the following discussion of the binding of 1 and its ana-
logues to actin we focus only on the most stable orientation
of the ligand in the binding site, that is, on the complex with
the lowest QM/MM energy after optimization (see Support-
ing Information for details). The computed binding site of
the 1/G-actin complex has a H-bond network similar but not
identical to that of the crystal structure (PDB ID: 1ESV).[6]
The backbone RMSD of the two structures is 1.2Š, with
the main deviations occurring for surface residues. However,
all tertiary structural features are maintained and the gener-
al overlap of the structures is good (Figure 8).
lin B (3)[9] which is inferior to latrunculin B (2) (Figure 7)
and significantly less effective than the best of our novel
analogues. Overall, the following ranking was observed:
Lat-A (1) ꢀ 44 > 42 ꢀ Lat-B (2) > 16-epi-Lat-B (3).
Comparison of the “latrunculins” bearing the natural 2-
thiazolidinone ring with their respective 2-oxazolidinone
anaACHTREUNGlogues reveals yet another important aspect. Although
somewhat less potent, all compounds of the latter series
retain significant bioactivity. This result corroborates the
notion that the presence of the sulfur atom is not essential
for actin binding as we had anticipated from the analysis of
the bonding situation observed in the 1/G-actin co-crystals
(see above). The best oxazolidinone-containing analogue 39
exhibits an activity that exceeds that of the natural product
16-epi-latrunculin B (3)[9] but is weaker than that of 1 or 2
(cf. Figure 7). Whether the slight decrease in potency in
going form the S- to the O-series is due to the larger size
and better polarizibility of the sulfur atom or to modulations
of other physicochemical properties remains yet to be eluci-
dated. What is even more remarkable, however, is the find-
ing that all 2-oxazolinone containing derivatives investigated
are markedly less cytotoxic than their relatives of the 2-thia-
zolidinone series. Specifically, 100% of the NIH3T3 fibro-
blasts were alive after 24 h of incubation with 10 mm solu-
tions of either 39 or 45, whereas even a concentration of
5 mm of 1 or 2 kills > 50% of the cultured cells during the
same time.
While the data outlined above indicate that the latruncu-
lins can accommodate substantial structural editing without
loss of their physiological properties and may even lead to
improved biological profiles, opening of the macrocycle is
largely detrimental. Thus, formal “cleavage” of the macro-
cylic edifice as realized in 50 and 51 leaves only weak actin-
binding properties behind. Likewise, the previously un-
known degradation product of latrunculin B, that is com-
pound 49,[11] did not induce any noticeable morphological
changes in the fibroblast cell culture.
Computational study: The biological activities of the differ-
ent analogues summarized above suggest a difference in
their binding abilities to G-actin. Therefore a computational
study was carried out to investigate the structure of the
binding site with bound latrunculin A (1) and consider the
changes that occur in the presence of the smaller homologue
latrunculin B (2) and the most potent of the fully synthetic
analogues, that is, compound 44. These three ligands show
various levels of activity (1 ꢀ 44 > 2) and the aim of this
study was to rationalize this experimental ranking.
The structure of actin with bound 1, available from the
protein data bank (PDB ID: 1ESV),[6] was used as the start-
ing point for the computational study. A conformational
search was performed for 1 and two of its analogues, 2 and
44, at the semi-empirical level of theory and the resulting
structures were subsequently optimized at the DFT level.
The conformers were then docked into the actin-binding site
Figure 8. Overlap of the crystal structure backbone (blue) and the opti-
mized 1/G-actin complex backbone (red).
The latrunculin A ligands from the optimized complex
and the crystal structure match well (Figure 9a). However,
the optimization of the binding site results in some changes
in the H-bond network relative to the crystal structure due
to missing side-chains. In the original PDB structure the
side-chain of Glu207 was not resolved, and thus an interac-
tion between the OH group of 1 and the NH group of
Arg210 was listed as a stabilizing interaction. In the opti-
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141

A. Fürstner et al.
Figure 9. a) Overlap of the crystal (blue) and optimized (red) latrunculin
A (1) residue in the binding site of G-actin; b) overlap of the residues
forming the binding site for the crystal (blue) and optimized (red) com-
plexes; the blue and red spheres represent oxygen atoms of water mole-
cules.
Figure 10. H-bond network of 1 (top) and 2 (bottom) in the actin-binding
site after the QM/MM optimization.
mized structure, the side-chain of Glu207 is present and its
carboxylate oxygen atom interacts strongly (rACTHRE(UNG H···O)=
while the macrocycle has no obvious H-bond pairing with
the protein.[38] In the head-group, the NH and CO moieties
form strong H-bonds to Asp157 and Thr186, respectively,
while there exists only a weak interaction with Lys213. The
O-atom of the tetrahydropyran ring forms a strong H-bond
with Tyr69.
The binding site of the 2/G-actin complex (Figure 10,
bottom) has a similar, but weaker H-bond network com-
pared with the complex formed by 1. Considering the 2-thia-
1.77 Š) with the NH of Arg210. The OH group of 1 is able
to form a strong hydrogen bond to one of the crystal waters,
which in turn binds to the carboxylate O atoms of Glu207.
There are also changes in the water positions around the
binding site (spheres in Figure 9b). However, these have no
effect on the H-bond network of the complex and are pre-
dominantly caused by a lack of strong H-bond partners
(e.g., outside the binding site).
Given the generally good over-
Table 2. Hydrogen bonds between the ligands and the actin-binding site; r = rH–Xacc refers to the bond length
[Š], a = aY-H-Xacc to the bond angle [8].
lap of the optimized and crys-
tallographic binding site struc-
tures, and the problems with
the unresolved side-chains in
the latter, we consider the com-
puted structure to be a reliable
representation of the complex.
In the optimized binding site
of the 1/G-actin complex
(Figure 10, top) there are
strong H-bonds between the
head-group of 1 and the sur-
Head-group
H-Bond[a]
1
2
44
r
a
r
a
r
a
NH···Asp157
CO···Thr186
CO···Lys213[b]
O1···Tyr69
1.58
1.73
–
1.94
–
176
167
–
163
–
1.63
–
–
2.00
2.01
159
–
–
156
146
1.60
–
1.73
1.98
–
175
–
157
154
–
OH···Glu207
[a] Criterion adopted for a hydrogen bond: rH–Xacc ꢄ 2.5 Š and aY-H-Xacc ꢀ 1408. The putative hydrogen bonds
forming the water bridge between the acyl group of latrunculin A and Glu214[10] are way beyond these thre-
AHCTREUNG
rounding residues (Table 2), ꢅ2.6 Š and aY-H-Xacc ꢅ1308).
142
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Chem. Eur. J. 2007, 13, 135 – 149

Latrunculin Analogues
FULL PAPER
zolidinone ring and the surrounding residues the strong NH
group binding to Asp157 persists, but the strong H-bond be-
tween the CO group and Thr186 is lost, with only the weak
interaction to Lys213 retained (Table 2). The H-bond be-
tween the oxygen atom of the tetrahydropyran ring and
Tyr69 is slightly weaker compared to the analogous H-bond
in the 1/G-actin complex; however, an additional H-bond
between the OH group and Glu207 exists in the 2/G-actin
complex, which will partially compensate for the other
losses. Overall, the result is a weaker H-bond network with
the two strong and one average H-bond in the 1/G-actin
complex being exchanged for one strong and two average
H-bonds in the 2/G-actin complex.
The individual binding energy of the substrates can in
principle be calculated directly. However, binding potential
energies generally overestimate the binding free energy of a
ligand to the receptor (which is measured experimentally)
due to the neglect of the dynamic factors that influence the
binding ability of ligands. Nonetheless, assuming that en-
tropic and desolvation effects are approximately equivalent
for the three ligands, the relative binding potential energies
(DDEbind) may be reasonable tools to compare binding abili-
ties between the ligands.[39] In fact, they could be calculated
as the difference between the relative stability of the ligand-
enzyme complexes (DEisoR) and of the free ligands in the
gas phase (DEisoL) (Scheme 9).
The H-bond network in the 44/G-actin complex binding
site (Figure 11) is different from the 1/G-actin complex but
is expected to be of a similar strength. The strong H-bond
between the carbonyl and Thr186 (Table 2) is lost; however,
this loss is offset by a strong ionic interaction with Lys213.
The other dominant H-bonds in the 1/G-actin complex
(NH···Asp157 and O1···Tyr69) are essentially unchanged.
Thus, although there is a shift in the binding pattern for the
44/G-actin complex, the qualitative strength of the H-bonds
(two strong and one average) remains similar to the parent
complex.
Scheme 9. Thermodynamic cycle for calculating relative binding energies.
DDEbind for the ligands can be determined from the homo-
desmotic reactions defined in Scheme 10. The 2/G-actin
complex is destabilized by 12.5 kcalmolꢁ1 and the 44/G-
actin complex by 7.5 (= 12.5–5.0) kcalmolꢁ1, relative to the
1/G-actin complex. Considering the isolated ligands, we find
that 2 and 44 are destabilized by 2.3 and 1.2 kcalmolꢁ1 rela-
Figure 11. H-bond network of 44 in the actin-binding site after the QM/
MM optimization.
The changes in the H-bond network hence offer a qualita-
tive explanation for the observed differences in the biologi-
cal activity of the three ligands. However, the contributions
of the hydrophobic effect will also aid in stabilizing the li-
gands in the binding site. In accordance with the published
crystal structure analysis,[6] the computational analysis shows
that only a part of the macrocycle of latrunculin A (1)
enjoys contacts to a hydrophobic environment, whereas the
rest is solvent exposed or surrounded by polar substituents.
Although the estimated hydrophobic contribution to the sta-
bility of the complex is higher for 1 than for its smaller con-
gener 2 and the synthetic analogue 44, no clear distinction
between the three ligands is possible (for details, see Sup-
porting Information).
Scheme 10. Computed homodesmotic reactions for comparing the stabili-
ty of 1 to 2, and 2 to 44. The partners in the isodesmic reactions (pent-2-
ene, hept-2,4-diene, nona-2,6-dienoic acid, and 3,8-dimethyl-2,6-dienoic
acid) were optimized in the gas phase (QM=MNDO/H).
Chem. Eur. J. 2007, 13, 135 – 149
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www.chemeurj.org
143

A. Fürstner et al.
tive to 1 in the gas phase. Using these values, the relative
binding energies decrease in the order 1 > 44 > 2 (0 vs 6.4
vs 10.2kcalmol ꢁ1).
provided that the conspicuous 2-thiazolidinone ring present
in all naturally occurring latrunculins is not essential for
actin binding and can be replaced by an oxazolidinone
moiety. Even though this engenders a slight loss in efficacy,
the cytotoxicity of the corresponding products is also re-
duced compared to their thiazolidinone analogues. Further-
more, the inversion of the absolute configuration of the
chiral center at C16 is well accommodated. Taken together,
these results clearly illustrate the power of “diverted total
synthesis”, as none of the relevant structural variations
could be achieved by derivatization of the natural leads
without undue preparative efforts. Finally, the relative po-
tency of three prototype members of this series (1 ꢀ 44 >
2) could be rationalized by a computational analysis of their
binding abilities to G-actin. This study revealed subtle dif-
ferences in the hydrogen bond network engaged by these li-
gands as well as distortion energies induced by the protein
host which offer a qualitative understanding of the experi-
mental data.
In all cases the ligand is destabilized upon binding to the
receptor due to the steric constraints imposed by the binding
site. The strain energy resulting from this distortion is calcu-
lated by comparing the energy of the ligand in its bound ge-
ometry and in the gas-phase optimized geometry. Latruncu-
lin A (1) and 44 are destabilized by similar amounts (15.2
and 16.8 kcalmolꢁ1, respectively), but less so than latruncu-
lin B (2, 21.5 kcalmolꢁ1).
In summary, the differences in the H-bond network, in
concert with the distortion energies of the ligands, offer a
qualitative understanding of the changes in the biological
activity for the individual compounds. Latrunculin A (1)
and 44 both have similar H-bond network strengths and
present similar ligand distortion. The hydrophobic effect,
however, slightly favors 1. Thus, we expect 1 and 44 to have
similar binding abilities to the receptor. In contrast, the H-
bond network is weaker for latrunculin B (2) and the distor-
tion of the ligand from its optimum geometry is larger in
this case. The hydrophobic contribution should slightly favor
2 over 44, although it is still lower than for 1. Thus, from
these structural arguments we expect that the binding ability
follows the order 1 ꢀ 44 > 2, which is in good agreement
with the experimental results.
The role of dynamic effects in influencing the binding
ability of 1 and its analogues, however, is an open question.
The optimized structures of the ligands bound to actin show
them to be partially buried in a hydrophobic pocket that
must open in order for the ligand to bind or unbind to the
receptor. This description is consistent with earlier reports
of inorganic phosphates binding in a region close to the
ligand-binding pocket, where it was found that significant
changes in subdomain 2of actin are required for binding the
phosphate.[40] A dynamic modulation of the binding mecha-
nism (conformation gating) has been shown previously to
affect the selectivity of some enzymes.[41,42] The role that
such a mechanism plays in the binding of 1 and its analogues
is the focus of current work.
Experimental Section
General methods: All reactions were carried out in flame-dried glassware
under Ar. The solvents were purified by distillation over the drying
agents indicated and were transferred under Ar: THF, Et2O, 1,4-dioxane
(Mg/anthracene), CH2Cl2 (P4O10), MeCN, Et3N, pyridine (CaH2), MeOH
(Mg), DMF (Desmodur, dibutyltin dilaurate), hexane, toluene (Na/K).
Flash chromatography: Merck silica gel 60 (230–400 mesh). NMR: Spec-
tra were recorded on a Bruker DPX 300, AV 400, or DMX 600 spectrom-
eter in the solvents indicated; chemical shifts (d) are given in ppm rela-
tive to TMS, coupling constants (J) in Hz. The solvent signals were used
as references and the chemical shifts converted to the TMS scale
ꢂ
ꢂ
(CDCl3: dC
CD2Cl2: dC
77.0 ppm; residual CHCl3 in CDCl3: dH
53.8 ppm; residual CH2Cl2 in CD2Cl2: dH
7.24 ppm;
5.32ppm).
ꢂ
ꢂ
Where indicated, the signal assignments are unambiguous; the numbering
Scheme is arbitrary and is shown in the inserts. The assignments are
based upon 1D and 2D spectra recorded using the following pulse se-
quences from the Bruker standard pulse program library: DEPT; COSY
(cosygs and cosydqtp); HSQC (invietgssi) optimized for 1J
ACHTREUNG
145 Hz; HMBC (inv4gslplrnd) for correlations via nJ
ACHTREUNG
TOCSY (invietgsml) using an MLEV17 mixing time of 120 ms. IR: Nico-
let FT-7199 spectrometer, wavenumbers (n˜) in cmꢁ1. MS (EI): Finnigan
MAT 8200 (70 eV), ESI-MS: Finnigan MAT 95, accurate mass determi-
nations: Bruker APEX III FT-MS (7 T magnet). Melting points: Büchi
melting point apparatus B-540 (corrected). Elemental analyses: H.
Kolbe, Mülheim/Ruhr. All commercially available compounds (Fluka,
Lancaster, Aldrich) were used as received.
Conclusion
The preparation of compounds 42–47, together with a compilation of
their analytical and spectroscopic data, can be found in ref. [13] (Support-
ing Information) and is therefore not repeated herein. The bare macrocy-
cle 52 is described in ref. [22b].
The largely catalysis-based and highly convergent total syn-
thesis of latrunculin A (1) and B (2) outlined in the accom-
panying paper was diverted to the preparation of a focused
library of analogues of these potent actin-binding macrolides
that enjoy widespread use in chemical biology. The chosen
approach allowed for structural variations of all characteris-
tic parts of the natural leads and hence enabled the first sys-
tematic mapping of the previously largely unknown struc-
ture/activity profile of this class of bioactive natural prod-
ucts. Thereby it was found that the removal of the methyl
branches decorating the macrocycle in 2 engenders a signifi-
cant increase in potency, while streamlining the synthesis to
a considerable extent. Moreover, compelling evidence is
Bioassay: Murine NIH/3T3 fibroblasts (CRL-1658 from ATCC) were
cultured at 378C and 5% CO2 in Dulbeccoꢁs modified Eagleꢁs medium
supplemented with 4 mm l-glutamine, 4.5 gLꢁ1 glucose and 10% bovine
calf serum. 2”104 cells were seeded on coverslips in one well of a 24-well
plate. After adapting and attaching over night, the cells were incubated
with 1 mm, 5 mm or 10 mm of the corresponding latrunculin compound for
18 h. Before and after each fixation or staining step the cells were
washed three times with TPBS (0.2% Tween20 in phosphate-buffered
saline). Cells were fixed with 3.7% formalin in PBS. For blocking unspe-
cific epitopes, fixed cells were incubated with 1% powdered milk in PBS.
Actin filaments were stained for 1 h with a solution of 77 nm TRITC la-
beled phalloidin (P1951, Sigma) in TPBS. Cell nuclei were stained with
144
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 135 – 149

Latrunculin Analogues
FULL PAPER
DAPI
(2-(4-amidinophenyl)-6-indolecarbamidine
dihydrochloride,
other portion of Et3N (0.58 mL, 4.2mmol). The mixture was stirred over-
night and was quenched with sat. aq. NH4Cl, the aqueous phase was ex-
tracted with CH2Cl2 (3”20 mL) and the combined organics were dried
(Na2SO4) and evaporated. The residue was purified by flash chromatog-
raphy (hexane/EtOAc 1:2) to give 24 as a light yellow oil (1.16 g, 99%).
D9542, Sigma). Cells were visualized and photographed with a Zeiss Axi-
ophot fluorescence microscope.
Methyl (4S)-2-oxo-1,3-oxazolidine-4-carboxylate (22):[43] Triethylamine
(13.4 mL, 96.3 mmol) was added at 08C over 5 min to a stirred suspen-
sion of serine methyl ester hydrochloride 21 (5.0 g, 32.1 mmol) in CH2Cl2
(75 mL). The reaction mixture was stirred for 10 min before a solution of
triphosgene (3.2g, 10.9 mmol) in CH 2Cl2 (25 mL) was added dropwise
over 2h. The reaction was stirred for 30 min, diluted with Et 2O (75 mL)
and cooled to ꢁ788C to precipitate all Et3NHCl salts. The mixture was
filtered and then concentrated to approx. 10 mL when it was applied
carefully to a 2.5 cm depth column of silica (prepacked EtOAc) in a
100 mL sinter funnel. The solution was washed through the column with
EtOAc (300 mL) and the filtrate was concentrated to give 22 as a color-
less oil (4.64 g, 99%). [a]D20 =ꢁ2.0 (c 0.62, EtOH); 1H NMR (300 MHz,
CDCl3): d=3.82(s, 3H), 4.43 (dd, J=9.5, 4.5 Hz, 1H), 4.57 (dd, J=8.9,
4.5 Hz, 1H), 4.60 (d, J=9.5 Hz, 1H), 6.38 (brs, 1H); 13C NMR (75 MHz,
CDCl3): d=53.2, 53.8, 66.8, 159.1, 170.6; IR (film): n˜ = 3296, 2959, 1732,
1
[a]2D0 =ꢁ4.5 (c 0.37, EtOH); H NMR (400 MHz, [D]6DMSO): d=3.12(s,
3H), 3.51 (s, 3H), 3.76 (s, 3H), 4.01 (d, J=14.9 Hz, 1H), 4.19 (dd, J=6.6,
2.2 Hz, 1H), 4.47–4.55 (m, 2H), 4.62 (d, J=14.9 Hz, 1H), 6.93 (appd, J=
8.7 Hz, 2H), 7.21 (appd, J=8.7 Hz, 2H); 13C NMR (100 MHz, CDCl3):
d=32.0, 45.7, 53.9, 54.9, 61.2, 64.2, 113.9, 127.6, 129.4, 157.6, 158.7, 169.0;
IR (film): n˜ = 3478, 2941, 2839, 1755, 1673, 1612, 1586, 1514, 1443, 1419,
1376, 1323, 1304, 1249, 1210, 1178, 1116, 1083, 1034, 997, 960, 846, 762,
559 cmꢁ1; MS (EI): m/z (%): 294 (7), 263 (23), 162 (6), 121 (100), 78 (6),
55 (7); HRMS (ESI): m/z: calcd for C14H18N2O5 +Na: 317.1113; found:
317.1112[ M ++Na].
(4S)-4-Acetyl-3-(4-methoxybenzyl)-1,3-oxazolidin-2-one (6): MeMgBr
(2.9 mL, 3m in Et2O, 8.6 mmol) was added at ꢁ408C to a solution of
compound 24 (2.4 g, 8.2 mmol) in THF (25 mL). The reaction was stirred
at ꢁ408C for 1 h and then allowed to warm to ꢁ208C for 2h. The reac-
tion was quenched with sat. aq. NH4Cl, the organic phase was separated
and the aqueous layer extracted with EtOAc (3”20 mL), the combined
organic phases were dried (Na2SO4) and evaporated, and the residue was
purified by flash chromatography (hexane/EtOAc 1:1) to give ketone 6
as a white solid (1.95 g, 96%). Chiral LC/MS showed that the product
had an ee of 77.3%. The material was recrystallized from tert-butyl
methyl ether to give white crystals (1.66 g, 82%, 97.2% ee). M.p. 65–
678C; [a]2D0 =ꢁ26.5 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d=2.07
(s, 3H), 3.81 (s, 3H), 4.06 (dd, J=9.8, 5.6 Hz, 1H), 4.11 (d, J=14.6 Hz,
1H), 4.13 (dd, J=8.9, 5.6 Hz, 1H), 4.43 (dd, J=9.8, 8.9 Hz, 1H), 4.80 (d,
J=14.6 Hz, 1H), 6.87 (appd, J=8.7 Hz, 2H), 7.16 (appd, J=8.7 Hz,
2H); 13C NMR (75 MHz, CDCl3): d=25.9, 47.1, 55.4, 62.4, 63.5, 114.4,
126.9, 130.1, 157.8, 159.7, 204.3; IR (film): n˜ = 3002, 2935, 2838, 1756,
1725, 1612, 1586, 1514, 1443, 1412, 1362, 1304, 1248, 1207, 1176, 1115,
1077, 1038, 846, 754 cmꢁ1; MS (EI): m/z (%): 249 (6), 206 (9), 121 (100),
91 (3), 78 (5), 77 (4); HRMS (ESI): m/z: calcd for C13H15NO4 +Na:
272.0898; found: 272.0895 [M ++Na].
Ethyl (Z)-3-chloro-2-propenoate (26):[44] A mixture of ethyl propiolate 25
(2.0 g, 20.4 mmol, 2.04 mL), dry lithium chloride (0.95 g) and acetic acid
(1.3 mL) in MeCN (25 mL) was refluxed for 18 h. The reaction was al-
lowed to cool and water added (100 mL). Solid K2CO3 was added until
no further CO2 evolution occurred. The organic layer was separated and
the aqueous phase extracted with tert-butyl methyl ether (3”100 mL).
The combined organic extracts were dried (Na2SO4) and evaporated. Pu-
rification of the residue by flash chromatography gave 26 as a colorless
liquid (1.66 g, 60%). 1H NMR (300 MHz, CDCl3): d=1.32(t, J=7.1 Hz,
3H), 4.25 (q, J=7.1 Hz, 2H), 6.20 (d, J=8.2Hz, 1H), 6.71 (d, J=8.2Hz,
1H); 13C NMR (100 MHz, CDCl3): d=163.4, 132.3, 121.5, 60.7, 14.2; MS
(EI): m/z (%): 106 (9), 99 (42), 91 (34), 89 (100), 61 (20), 45 (14); HRMS
(CI, isobutane): m/z: calcd for C5H7ClO2: 135.0212; found: 135.0210 [M +
+H].
1480, 1438, 1399, 1366, 1213, 1115, 1056, 1007, 954, 920, 825, 766 cmꢁ1
MS (EI): m/z (%): 145 (7), 86 (100), 58 (11), 42(55); HRMS (EI): m/z:
calcd for C5H7NO4: 145.0375; found: 145.0372.
;
Methyl
(4S)-3-(4-methoxybenzyl)-2-oxo-1,3-oxazolidine-4-carboxylate
(23): A solution of 22 (3.5 g, 24.1 mmol) in THF (30 mL) was slowly
added at ꢁ158C to a slurry of NaH (0.61 g, 25.3 mmol) in THF (50 mL).
The reaction was allowed to stir for 3 h before a solution of p-methoxy-
benzyl bromide (PMBBr, 8.5 g, 42mmol) in THF (20 mL) was added
dropwise. The reaction was stirred for 16 h before it was quenched with
aq. sat. NH4Cl (40 mL). The aqueous phase was extracted with tert-butyl
methyl ether (2”40 mL), the combined organic layers were dried
(Na2SO4) and evaporated, and the residue was purified by flash chroma-
tography (hexane/EtOAc 3:1) to give product 23 as a white solid (3.57 g,
56%). M.p. 65–678C; [a]2D0 =ꢁ25.0 (c 0.45, EtOH); 1H NMR (300 MHz,
CDCl3): d=3.76 (s, 3H), 3.81 (s, 3H), 4.09 (dd, J=9.4, 5.1 Hz, 1H), 4.18
(d, J=14.7 Hz, 1H), 4.32(dd, J=9.0, 5.1 Hz, 1H), 4.38 (dd, J=9.4,
9.0 Hz, 1H), 4.84 (d, J=14.7 Hz, 1H), 6.87 (appd, J=8.6 Hz, 2H), 7.19
(appd, J=8.6 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=46.8, 52.9, 55.4,
55.9, 64.4, 114.3, 127.1, 130.0, 157.6, 159.6, 170.0; IR (film): n˜ = 2973,
2959, 1732, 1613, 1585, 1515, 1466, 1435, 1415, 1364, 1316, 1302, 1285,
1245, 1209, 1174, 1113, 1086, 1048, 1025, 1009, 980, 966, 946, 921, 870,
837, 828, 814, 756, 744, 715, 671 cmꢁ1; MS (EI): m/z (%): 265 (28), 179
(30), 162(14), 135 (16), 134 (37), 121 (100), 78 (11); HRMS (EI): m/z:
calcd for C13H15NO5: 265.0950; found: 265.0952.
(4S)-N-Methoxy-3-(4-methoxybenzyl)-N-methyl-2-oxo-1,3-oxazolidine-4-
carboxamide (24): Aqueous KOH (2.15 g in 35 mL of H2O, 38 mmol) was
added to a solution of 23 (3.38 g, 12.7 mmol) in 1,4-dioxane (50 mL).
After 1 h the reaction was acidified with aq. HCl (2m) and diluted with
tert-butyl methyl ether (200 mL). The aqueous layer was extracted with
tert-butyl methyl ether (2”100 mL), the combined organic phases were
washed with brine, dried (Na2SO4) and concentrated to an oil. Repeated
treatment of the oil with toluene/CH2Cl2 2:1 followed by re-evaporation
resulted in removal of residual water and isolation of (4S)-3-(4-methoxy-
benzyl)-2-oxo-1,3-oxazolidine-4-carboxylic acid as a white solid (3.1 g,
97%). M.p. 116–1188C; [a]D20 =ꢁ28.1 (c 1.0, EtOH); 1H NMR (300 MHz,
[D]6DMSO): d=3.76 (s, 3H), 4.09 (d, J=15.1 Hz, 1H), 4.14 (dd, J=9.4,
4.4 Hz, 1H), 4.29 (dd, J=8.9, 4.4 Hz, 1H), 4.47 (t, J=9.4 Hz, 1H), 4.62
(d, J=15.1 Hz, 1H), 6.93 (appd, J=8.7 Hz, 2H), 7.21 (appd, J=8.7 Hz,
2H); 13C NMR (75 MHz, CDCl3): d=45.8, 55.1, 55.8, 64.5, 114.0, 127.7,
129.4, 157.3, 158.8, 171.3; IR (film): n˜ = 3440, 2958, 2934, 2838, 2722,
2611, 2509, 1743, 1689, 1612, 1587, 1514, 1470, 1444, 1419, 1365, 1294,
Ethyl (Z)-2-octen-6-ynoate (28): Magnesium turnings (433 mg,
17.8 mmol) were stirred with I2 for 1 h. THF (2mL) was added and a so-
lution of 5-pentynyl bromide (2.3 g, 16.0 mmol) in THF (17 mL) was
added dropwise to maintain gentle reflux. After complete addition, the
mixture was refluxed for 1.5 h before allowing to cool. The solution of
the Grignard reagent 27 thus formed was quickly added in one portion to
a solution of chloride 26 (1.0 g, 7.4 mmol) and [FeCAHTRE(UNG acac)3] (565 mg,
10 mol%) in THF (15 mL) at ꢁ308C. After 10 min the reaction was al-
lowed to warm to room temperature before quenching with sat. aq.
NH4Cl. The organic layers were separated and the aqueous phase was ex-
tracted with tert-butyl methyl ether. The combined organic layers were
dried (Na2SO4), the solvent was evaporated and the residue purified by
flash chromatography (hexane/EtOAc 20:1) to give 28 as a volatile light
yellow oil (964 mg, 78%). 1H NMR (300 MHz, CDCl3): d=1.28 (t, J=
7.1 Hz, 3H), 1.77 (t, J=2.5 Hz, 3H), 2.27 (m, 2H), 2.82 (dd, J=7.2,
1.7 Hz, 2H), 4.16 (q, J=7.1 Hz, 2H), 5.81 (ddd, J=1.7, 1.7, 11.4 Hz, 1H),
6.32(dd, J=7.2, 11.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=3.5, 14.3,
18.4, 28.4, 59.9, 76.4, 78.0, 120.7, 148.3, 166.3; IR (film): n˜ = 2981, 2920,
1268, 1248, 1197, 1179, 1116, 1101, 1030, 971, 834, 813, 762, 746, 677 cmꢁ1
MS (EI): m/z (%): 251 (58), 206 (5), 179 (81), 162 (15), 134 (94), 121
(100), 78 (16), 77 (15); HRMS (EI): m/z: calcd for C12H13NO5: 251.0793;
found: 251.0794.
;
To a solution of this acid (1.0 g, 4 mmol) in CH2Cl2 (10 mL) was added
(benzotriazol-1-yloxy) tris-(dimethylamino)-phosphonium hexafluoro-
phosphate (BOP, 1.8 g, 4 mmol) shortly followed by Et3N (0.58 mL,
4.2mmol). The reaction was stirred for 10 min before N,O-dimethylhy-
droxylamine hydrochloride (0.43 g, 4.4 mmol) was added along with an-
Chem. Eur. J. 2007, 13, 135 – 149
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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145

A. Fürstner et al.
1716, 1646, 1445, 1414, 1388, 1332, 1283, 1216, 1188, 1163, 1096, 1030,
821 cmꢁ1; HRMS (CI, isobutane): m/z: calcd for C10H15O2: 167.1072;
found: 167.1073 [M ++H].
in CH2Cl2, 1.7 mL, 1.7 mmol) was introduced. After stirring for 2h at
ꢁ788C, the reaction was warmed to 08C and allowed to stir for 3 h. The
reaction was cooled to ꢁ788C and a solution of aldehyde 14 (250 mg,
1.04 mmol) in CH2Cl2 (5 mL) was added dropwise. After stirring for 3 h
at ꢁ788C, the reaction was quenched with aq. NH4Cl and the mixture al-
lowed to warm to room temperature before water was added to dissolve
the salts. The organic layer was separated and the aqueous phase extract-
ed with CH2Cl2 (3”20 mL).
(Z)-2-Octen-6-ynoic acid (9): Aq. NaOH (1m, 12.4 mL, 12.4 mmol) was
added to a stirred solution of ester 28 (773 mg, 4.65 mmol) in MeOH
(10 mL). The reaction was allowed to stir overnight before removal of
the methanol under vacuum. The aqueous layer was washed with tert-
butyl methyl ether and the organic phases were discarded. The aqueous
layer was acidified to pH 1 with aq. HCl (2m) and extracted with CH2Cl2
(3”20 mL). The combined organic phases were dried (Na2SO4) and
evaporated and the residue was purified by Kugelrohr distillation to give
9 as a colorless liquid (571 mg, 74%). 1H NMR (300 MHz, CDCl3): d=
1.77 (t, J=2.5 Hz, 3H), 2.23–2.32 (m, 2H), 2.82 (dd, J=7.1, 1.7 Hz, 2H),
5.84 (ddd, J=11.5, 1.6, 1.6 Hz, 1H), 6.44 (ddd, J=11.5, 7.1, 7.1 Hz, 1H),
11.5 (brs, 1H); 13C NMR (100 MHz, CDCl3): d=3.5, 18.4, 28.7, 76.7,
77.7, 120.0, 151.3, 171.8; IR (film): n˜ = 2970, 2921, 1737, 1695, 1432,
1366, 1287, 1231, 1217, 1204, 1110, 924, 826, 722 cmꢁ1; HRMS (CI, isobu-
tane): m/z: calcd for C8H11O2: 139.0759; found: 139.0760 [M ++H].
The combined organic layers were washed with aq. NaHCO3 and brine,
dried (Na2SO4) and concentrated. The residue was purified by flash chro-
matography (hexane/EtOAc 2:1) to give the product in form of two dia-
stereomers that were separable by chromatography (hexane/EtOAc 3:1).
Major isomer 32: Colorless oil (163 mg, 30%); [a]2D0 =ꢁ4.4 (c 0.73,
1
CHCl3); H NMR (400 MHz, CDCl3): d=0.09 (d, J=4.6 Hz, 6H), 0.87 (s,
9H), 1.37–1.61 (m, 6H), 1.74 (t, J=2.5 Hz, 3H), 2.07–2.14 (m, 2H), 2.21
(dd, J=15.3, 3.7 Hz, 1H), 2.51 (dd, J=15.3, 8.7 Hz, 1H), 3.51 (brs, 1H),
3.77 (s, 3H), 3.90–3.97 (m, 1H), 4.02(d, J=14.7 Hz, 1H), 4.08–4.15 (m,
1H), 4.19 (m, 2H), 4.35 (m, 1H), 4.73 (d, J=14.7 Hz, 1H), 6.81 (appd,
J=8.6 Hz, 2H), 7.13 (appd, J=8.6 Hz, 2H); 13C NMR (100 MHz,
CDCl3): d=ꢁ4.8, ꢁ4.1, 3.3, 17.8, 18.6, 23.9, 25.7, 36.6, 42.4, 46.6, 46.8,
55.1, 62.4, 63.0, 67.7, 72.3, 75.9, 78.5, 114.1, 127.1, 130.0, 157.8, 159.5,
205.7; IR (film): n˜ = 3462, 2941, 2929, 2857, 1747, 1612, 1514, 1412, 1371,
1303, 1248, 1176, 1075, 1032, 835, 809, 774, 731, 663 cmꢁ1; MS (EI): m/z
(%): 460 (1), 442 (6), 211 (4), 206 (3), 169 (3), 167 (3), 122 (8), 121 (100),
101 (9), 93 (7), 91 (2), 75 (9), 73 (7), 59 (4); HRMS (ESI): m/z: calcd for
C28H43NO6Si: 518.2937; found: 518.2935 [M ++H].
(1R)-1-Allyl-5-heptynyl tert-butyl
5-heptynal 29 (1.4 g, 12.7 mmol) in diethyl ether (20 mL) was added at
ꢁ1008C to a stirred solution of (ꢁ)-Ipc2B
(allyl)[45] (25.4 mL, 0.5m in
ACHTRE(UNG dimethyl)silyl ether (31): A solution of
AHCTREUNG
Et2O, 12.7 mmol). The reaction was allowed to stir for 1 h at ꢁ1008C and
then quenched with MeOH (1 mL). The solvent was removed under
vacuum at 08C and the residue dissolved in MeOH (26 mL). 8-Hydroxy-
quinoline (2.3 g) was added and the reaction stirred overnight. The
yellow precipitate formed was filtered off and the filtrate evaporated to
dryness. Purification of the residue by flash chromatography (hexane/
EtOAc 40:1) gave slightly impure alcohol 30 as a light brown oil (1.93 g;
91% ee, GC) which was directly used in the next step.
Minor isomer 33: White solid (147 mg, 28%); m.p. 77–798C; [a]D20
=
+33.6 (c 0.78, CHCl3); 1H NMR (400 MHz, CDCl3): d=0.09 (s, 6H),
0.88 (s, 9H), 1.45 (m, 2H), 1.50 (ddd, J=14.3, 5.2, 2.2 Hz, 1H), 1.58–1.73
(m, 3H), 1.76 (t, J=2.5 Hz, 3H), 2.10–2.16 (m, 2H), 2.31 (dd, J=15.9,
3.7 Hz, 1H), 2.49 (dd, J=15.9, 8.5 Hz, 1H), 3.63 (brs, 1H), 3.78 (s, 3H),
3.95–4.01 (m, 1H), 4.04 (d, J=14.7 Hz, 1H), 4.11 (dd, J=9.6, 5.6 Hz,
The crude material was dissolved in DMF (20 mL) followed by the addi-
tion of TBSCl (2.4 g, 15.88 mmol) and imidazole (1.7 g, 25.4 mmol). The
reaction was stirred overnight before it was diluted with hexane
(250 mL). The solution was successively washed with 5% aq. HCl, sat.
aq. NaHCO3, water and brine followed by drying (Na2SO4) of the organic
phase and evaporation of the solvent. The residue was purified by flash
chromatography (hexane/EtOAc 40:1) to give product 31 as a colorless
oil (2.05 g, 68% over both steps). [a]2D0 =+9 (c 0.07, CHCl3); 1H NMR
(300 MHz, CDCl3): d=0.06 (s, 6H), 0.89 (s, 9H), 1.53 (m, 4H), 1.78 (t,
J=2.5 Hz, 3H), 2.12 (m, 2H), 2.22 (ddd, J=7.1, 5.9, 1.2Hz, 2H), 3.72
(m, 1H), 5.02(s, 1H), 5.06 (m, 1H), 5.82(m, 1H); 13C NMR (75 MHz,
CDCl3): d=ꢁ4.4, ꢁ4.3, 3.6, 18.2, 18.9, 24.9, 26.0, 36.0, 41.9, 71.8, 75.7,
79.3, 116.8, 135.4; IR (film): n˜ = 2952, 2929, 2857, 1472, 1462, 1435, 1361,
1254, 1088, 1005, 938, 911, 880, 834, 808, 772, 665 cmꢁ1; MS (EI): m/z
(%): 225 (4), 209 (5), 185 (3), 133 (14), 99, (22), 93, (44), 75 (100), 73
(67); HRMS (CI, isobutane): m/z: calcd for C16H30OSi: 267.2144; found:
267.2141 [M ++H].
1H), 4.19 (dd, J=8.9, 5.6 Hz, 1H), 4.34–4.42(m, 2H), 4.83 (d,
J=
14.7 Hz, 1H), 6.84 (appd, J=8.6 Hz, 2H), 7.12 (appd, J=8.6 Hz, 2H);
13C NMR (100 MHz, CDCl3): d=ꢁ4.8, ꢁ4.6, 3.4, 17.9, 18.7, 25.0, 25.7,
35.2, 41.1, 46.7, 47.2, 55.2, 62.0, 63.3, 64.9, 70.8, 76.0, 78.6, 114.2, 127.0,
110.0, 157.9, 159.5, 205.9; IR (KBr): n˜ = 3415, 2952, 2932, 2857, 1723,
1613, 1516, 1451, 1433, 1347, 1307, 1256, 1225, 1185, 1102, 1069, 1032,
1018, 941, 930, 838, 823, 808, 767, 753, 740, 726, 660 cmꢁ1; MS (EI): m/z
(%): 499 (0.8), 442 (11), 249 (6), 206 (8), 169 (8), 121 (100), 101 (12);
HRMS (ESI): m/z: calcd for C28H43NO6Si+Na: 540.2757; found:
540.2751 [M ++Na].
(4R)-4-[(2R,4S,6R)-6-(4-Hexynyl)-4-hydroxy-2-methoxytetrahydro-2H-
pyran-2-yl]-3-(4-methoxybenzyl)-1,3-oxazolidin-2-one (34): Aq. HCl (1m,
1.6 mL) was added to a stirred solution of aldol 33 (315 mg, 0.61 mmol)
in THF (12mL). The reaction was stirred overnight
(3R)-3-{[tert-ButylACHTREUNG(dimethyl)silyl]oxy}-7-nonynal (14): A stirred solution
and then quenched with aq. NaHCO3, the aqueous
layer was extracted with CH2Cl2 (4”20 mL), the com-
bined organic phases were washed with sat. aq. NaCl
and dried (Na2SO4). After evaporation of the solvent
the residue was purified by flash chromatography
(hexane/EtOAc 1:1) to give the corresponding hemia-
cetal which was used without delay in the next reac-
of compound 31 (2.01 g, 8.43 mmol) and trace amounts of Sudan Red 7B
(enough to give red color) in MeOH (100 mL) at ꢁ788C was treated
with ozone until the red color disappeared. Argon was bubbled through
the solution for 15 min before PPh3 (3.3 g, 1.5 equiv) was added and the
reaction left to stir overnight. The solvent was evaporated and the resi-
due purified by flash chromatography (hexane ! hexane/EtOAc 20:1) to
give aldehyde 14 as a colorless oil (1.51 g, 67%). [a]2D0 =ꢁ7.8 (c 0.7,
CHCl3); 1H NMR (300 MHz, CDCl3): d=0.07 (s, 3H), 0.09 (s, 3H), 0.88
(s, 9H), 1.46–1.70 (m, 6H), 1.78 (t, J=2.5 Hz, 3H), 2.15 (dddd, J=9.2,
5.1, 2.5, 2.4 Hz, 2H), 2.52 (dd, J=2.4, 1.5 Hz, 1H), 2.54 (t, J=2.4 Hz,
tion.
A catalytic amount of camphorsulfonic acid
(CSA) was added to a solution of the crude hemiace-
tal in methanol (4 mL). The reaction was stirred over-
night before quenching with sat. aq. NaHCO3. The
aqueous phase was extracted with CH2Cl2 (3”5 mL),
1H), 4.23 (tt, J=5.7 Hz, 1H), 9.82(t,
J=2.5 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d=ꢁ4.3, ꢁ4.0, 18.4, 19.1, 24.9, 26.1, 37.2, 51.1, 68.2,
76.4, 77.6, 79.0, 202.6; IR (film): n˜ = 2954, 2930, 2858, 2712, 1713, 1472,
1463, 1436, 1409, 1389, 1376, 1361, 1295, 1256, 1217, 1098, 1027, 1006,
939, 837, 811, 776, 680, 665 cmꢁ1; MS (EI): m/z (%): 211 (50), 169 (41),
167 (32), 157 (13), 129 (10), 119 (19), 101 (100), 93 (56), 75 (64), 59 (39);
HRMS (CI, isobutane): m/z: calcd for C15H28O2Si: 269.1936; found:
269.1933 [M ++H].
the combined organic layers were washed with brine,
dried (Na2SO4) and evaporated. The residue was puri-
fied by flash chromatography (hexane/EtOAc 2:1) to give glycoside 34
(77 mg, 92%) as a colorless oil. [a]2D0 =+30.6 (c 0.41, CHCl3); 1H NMR
(400 MHz, CDCl3): d=1.38–1.50 (m, 2H), 1.51–1.62 (m, 3H), 1.65 (dd,
J=14.4, 3.7 Hz, 1H), 1.73 (t, J=2.5 Hz, 3H), 1.77–1.88 (m, 2H), 2.07–
2.13 (m, 2H), 3.07 (s, 3H), 3.64 (brs, 1H), 3.78 (s, 3H), 3.81 (dd, J=9.3,
4.7 Hz, 1H), 3.83–3.89 (m, 1H), 4.12–4.26 (m, 4H), 4.94 (d, J=15.2Hz,
1H), 6.86 (appd, J=8.5 Hz, 2H), 7.17 (appd, J=8.5 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d=3.4, 18.8, 24.9, 32.5, 34.6, 37.4, 46.2, 47.9, 53.8,
Titanium aldol reaction: TiCl4 (1m in CH2Cl2, 1.35 mL, 1.35 mmol) was
added at ꢁ788C to a solution of ketone 7 (311 mg, 1.25 mmol) in CH2Cl2
(5 mL). The reaction was allowed to stir for 20 min before (iPr)2NEt (1m
146
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Chem. Eur. J. 2007, 13, 135 – 149

Latrunculin Analogues
FULL PAPER
55.3, 63.6, 64.2, 65.2, 75.9, 78.5, 102.1, 114.2, 128.1, 128.9, 158.9, 159.3; IR
(film): n˜ = 3520, 2942, 2838, 1745, 1612, 1585, 1513, 1439, 1410, 1366,
1303, 1287, 1229, 1175, 1103, 1080, 1026, 957, 886, 819, 766, 751, 735, 717,
675 cmꢁ1; MS (EI): m/z (%): 417 (0.3), 211 (53), 193 (8), 179 (19), 161
(15), 151 (9), 138 (7), 137 (80), 121 (100), 119 (56), 111 (17), 109 (10), 95
(14), 93 (10), 91 (13); HRMS (ESI): m/z: calcd for C23H31NO6 +Na:
440.2049; found: 440.2046 [M ++Na].
(d, J=11.7 Hz, 1H), 5.35–5.26 (m, 1H), 4.95–4.82 (m, 2H), 4.30–4.15 (m,
3H), 3.87 (dd, J=9.2, 4.7 Hz, 1H), 3.79 (s, 3H), 3.50–3.35 (m, 1H), 3.14
(s, 3H), 2.51–2.35 (m, 2H), 2.27–2.16 (m, 2H), 2.03 (d, J=14.7 Hz, 2H),
1.77–1.61 (m, 4H), 1.59–1.33 (m, 3H); 13C NMR (75 MHz, CDCl3): d=
166.0, 159.2, 159.0, 146.1, 129.0, 128.5, 122.8, 114.1, 100.2, 81.6, 80.9, 66.8,
64.9, 64.3, 55.3, 54.0, 48.0, 46.2, 35.9, 34.1, 30.7, 28.4, 22.2, 19.6, 19.0; IR
(film): n˜ = 2936, 1750, 1701, 1513 cmꢁ1; HRMS (ESI): m/z: calcd for
C27H33NO7 +Na: 506.2155; found: 506.2159 [M ++Na].
(4R)-4-[(2R,4R,6R)-6-(4-hexynyl)-4-hydroxy-2-methoxytetrahydro-2H-
pyran-2-yl]-3-(4-methoxybenzyl)-1,3-oxazolidin-2-one (35): Prepared
analogously from aldol 32; colorless oil (58 mg, 88%); [a]2D0 =+30.0
(c 0.41, CHCl3); 1H NMR (400 MHz, CDCl3): d=1.20
(m, 1H), 1.27–1.35 (m, 1H), 1.39–1.49 (m, 1H), 1.52–
1.61 (m, 3H), 1.74 (t, J=2.5 Hz, 3H), 1.92–2.03 (m,
2H), 2.07–2.13 (m, 2H), 2.53 (brs, 1H), 2.97 (s, 3H),
3.47–3.55 (m, 1H), 3.79 (s, 3H), 3.85 (dd, J=7.3,
6.8 Hz, 1H), 4.03 (dddd, J=10.4, 10.3, 4.3, 4.2Hz,
1H), 4.18–4.26 (m, 3H), 4.92 (d, J=15.3 Hz, 1H), 6.86
(appd, J=8.6 Hz, 2H), 7.18 (appd, J=8.6 Hz, 2H);
13C NMR (100 MHz, CDCl3): d=3.4, 18.8, 24.9, 34.7,
36.9, 40.1, 46.1, 47.7, 53.5, 55.3, 64.3, 69.8, 75.9, 78.6,
101.8, 114.2, 128.4, 128.9, 159.2; IR (film): n˜ = 3442,
2970, 2946, 1739, 1612, 1513, 1440, 1413, 1365, 1229,
1217, 1175, 1141, 1113, 1082, 1022, 963, 819, 751 cmꢁ1; MS (EI): m/z (%):
417 (0.5), 211 (51), 193 (7), 179 (12), 161 (21), 151 (7), 137 (64), 133 (43),
121 (100), 119 (60), 111 (18), 109 (13), 103 (15), 95 (34), 93 (11), 91 (15),
71 (11); HRMS (ESI): m/z: calcd for C23H31NO6 +Na: 440.2049; found:
440.2046 [M ++Na].
Compound 39: Commercial Lindlar catalyst (15 mg) was added to a solu-
tion of cycloalkyne 38 (9.1 mg, 18.8 mmol) in CH2Cl2 (2mL) and the re-
sulting mixture was stirred vigorously under a hy-
drogen atmosphere (1 atm) overnight. For work up,
the mixture was filtered through Celite and the fil-
trate was evaporated to give analytically pure (+)-
(R)-3-(4-methoxybenzyl)-4-((1R,4Z,8Z,13R,15R)-
15-methoxy-3-oxo-2,14-dioxa-bicyclo-
AHCTRE[UNG 11.3.1]heptadeca-4,8-dien-15-yl)oxazolidin-2-one
as a pale yellow oil (9.1 mg, quant.). [a]2D0 =+53.3
(c 1.09, CH2Cl2); 1H NMR (400 MHz, CDCl3): d=
7.20 (d, J=8.3 Hz, 2H), 6.86 (d, J=8.7 Hz, 2H),
6.27–6.15 (m, 1H), 5.81 (d, J=11.7 Hz, 1H), 5.35–
5.26 (m, 1H), 4.95–4.82 (m, 2H), 4.30–4.15 (m, 3H), 3.87 (dd, J=9.2,
4.7 Hz, 1H), 3.79 (s, 3H), 3.50–3.35 (m, 1H), 3.14 (s, 3H), 2.51–2.35 (m,
2H), 2.27–2.16 (m, 2H), 2.03 (d, J=14.7 Hz, 2H), 1.77–1.61 (m, 4H),
1.59–1.33 (m, 3H); 13C NMR (75 MHz, CDCl3): d=166.0 (C), 159.2(C),
159.0 (C), 145.5 (CH), 129.5 (CH), 129.0 (CH), 128.5 (C), 122.0 (CH),
114.1 (CH), 100.4 (C), 66.9 (CH), 64.2(CH 2), 62.9 (CH), 55.3 (CH), 54.3
(CH3), 47.9 (CH3), 46.2(CH 2), 34.1 (CH2), 30.8 (CH2), 30.8 (CH2), 30.2
(CH2), 27.3 (CH2), 23.7 (CH2), 21.8 (CH2); IR (film): n˜ = 2935, 1753,
Ester 36: Triflic anhydride (27 mL, 0.16 mmol) was added to a cooled
(ꢁ788C) solution of compound 34 (56.6 mg, 0.136 mmol) and pyridine
(22 mL, 0.27 mmol) in CH2Cl2 (5 mL). The mixture was warmed to
ꢁ458C and stirred at that temperature for 4 h. For work up, the reaction
was quenched with aq. KHSO4 (15 mL, 10% w/w) and ice, the aqueous
layer was repeatedly extracted with CH2Cl2, the combined organic phases
were dried over Na2SO4, filtered and evaporated. The resulting triflate
was dissolved in THF (7.5 mL) and added to a cooled solution (08C) of
NaH (15.6 mg, 0.390 mmol) and acid 9 (59.3 mmol, 0.429 mmol) in THF
(5 mL) [which had been stirred for 30 min at ambient temperature and
for additional 30 min at reflux temperature prior to use]. The resulting
mixture was stirred for 15 h at ambient temperature before it was poured
into sat. aq. NaHCO3. The aqueous layer was extracted with EtOAc (3”
10 mL), the combined organic phases were dried (Na2SO4) and evaporat-
ed, and the residue was purified by flash chromatography (hexanes/
1707, 1513 cmꢁ1
508.2311; found: 508.2308 [M ++Na].
; HRMS (ESI): m/z: calcd for C27H35NO7 + Na:
CAN (26 mg, 0.047 mmol) was added to a vigorously stirred suspension
of the cycloalkene prepared above (9.1 mg, 18.7 mmol) in MeCN/water
2:1 (0.6 mL). After 20 min the mixture became homogeneous and stirring
was continued for additional 21 h. The solution was extracted with
CH2Cl2 (3”2mL), the combined organic layers were dried (Na 2SO4) and
evaporated, and the residue was purified by flash chromatography
(hexane/EtOAc 1:1) to give product 39 as a colorless oil (6.1 mg, 93%).
[a]2D0 =+58.1 (c 1.1, CHCl3); 1H NMR (400 MHz, CD2Cl2): d=8.51 (s,
1H), 6.28–6.20 (m, 1H), 5.75 (d, J=11.6 Hz, 1H), 5.51 (dd, J=6.1,
2.0 Hz, 1H), 5.42–5.33 (m, 1H), 5.31–5.20 (m, 1H), 4.36 (t, J=9.0 Hz,
1H), 4.34–4.27 (m, 1H), 4.24 (dd, J=9.1, 6.1 Hz, 1H), 3.98–3.84 (m, 2H),
3.79 (ddd, J=9.0, 5.9, 1.3 Hz, 1H), 3.33 (d, J=1.0 Hz, 1H), 2.51–1.20 (m,
12H); 13C NMR (100 MHz, CD2Cl2): d=165.9 (C), 159.7 (C), 145.6
(CH), 129.8 (CH), 129.8 (CH), 121.8 (CH), 96.4 (C), 68.3 (CH), 66.1
(CH2), 62.5 (CH), 60.6 (CH), 35.4 (CH2), 32.6 (CH2), 31.5 (CH2), 31.0
(CH2), 27.6 (CH2), 24.2 (CH2), 22.4 (CH2); IR (film): n˜ = 3337, 2924,
EtOAc 2:1) to afford diyne 36 as a colorless oil (51.5 mg, 70%). [a]D20
=
+57.7 (c 1.02, CHCl3); 1H NMR (400 MHz, CDCl3): d=7.19 (d, J=
8.6 Hz, 2H), 7.00 (dt, J=15.7, 6.6 Hz, 1H), 6.85 (d, J=8.6 Hz, 2H), 5.88
(dt, J=15.7, 1.5 Hz, 1H), 5.19 (m, 1H), 4.88 (d, J=15.2Hz, 1H), 4.25–
4.08 (m, 2H), 4.21 (d, J=15.2Hz, 1H), 3.91–3.83 (m, 1H), 3.83–3.77 (m,
1H), 3.79 (s, 3H), 3.07 (s, 3H), 2.44–2.37 (m, 2H), 2.35–2.27 (m, 2H),
2.15–2.08 (m, 2H), 1.96 (dt, J=15.2, 1.9 Hz, 1H), 1.86–1.79 (m, 1H), 1.78
(t, J=2.5 Hz, 3H), 1.74 (t, J=2.5 Hz, 3H), 1.64 (dd, J=15.2, 4.3 Hz,
2H), 1.57–1.41 (m, 4H); 13C NMR (100 MHz, CDCl3): d=165.7 (C),
159.2 (C), 159.0 (C), 147.4 (CH), 129.0 (CH), 128.5 (C), 122.5 (CH),
114.1 (CH), 99.9 (C), 78.5 (C), 77.5 (C), 76.6 (C), 75.9 (C), 66.6 (CH),
65.3 (CH), 64.2(CH 2), 55.2(CH 3), 54.2(CH), 47.8 (CH 3), 46.2(CH 2),
34.6 (CH2), 34.2(CH 2), 31.7 (CH2), 30.7 (CH2), 24.9 (CH2), 18.8 (CH2),
17.9 (CH2), 3.4 (CH3), 3.4 (CH3); IR (film): n˜ = 2920, 1749, 1709, 1513,
2858, 1747, 1706 cmꢁ1
.
Compound 48: Cerium ammonium nitrate (CAN, 26 mg, 0.047 mmol)
was added to a vigorously stirred suspension of cycloalkyne 53 (10 mg,
0.019 mmol)[11, 14a] in MeCN/water 2:1 (0.5 mL). After 20 min the mixture
became homogeneous and stirring was continued for additional 4 h. The
solution was extracted with CH2Cl2 (3”2mL), the combined organic
layers were dried (Na2SO4) and the solvent was evaporated. Purification
by flash chromatography (EtOAc/hexane 1:2) afforded derivative 48 as a
pale yellow oil (5 mg, 80%). 1H NMR (600 MHz, CDCl3): d=5.74 (s, J=
1.2Hz, 1H, H2), 5.68 (s, 1H, -NH), 5.35 (quint., J=2.9 Hz, 1H, H13),
4.71 (ddt, J=11.6, 6.8, 1.8 Hz, 1H, H11), 3.80
1030 cmꢁ1
; HRMS (ESI): m/z: calcd for C31H39NO7 +Na: 560.2624;
found: 560.2621 [M ++Na].
Cycloalkyne 38: CH2Cl2 (245 mL, 3.81 mmol) was added to a solution of
complex 40 (16 mg, 0.026 mmol)[22] in degassed toluene (2mL). After stir-
ring for 15 min, the resulting mixture was added to a solution of diyne 36
(51.5 mg, 0.096 mmol) in toluene (48 mL) and the resulting suspension
was stirred at 808C for 15 h. The mixture was then allowed to cool to am-
bient temperature, filtered through a pad of silica, and the filtrate was
evaporated. Purification of the residue by flash chromatography (hex-
anes/EtOAc 2:1) afforded cycloalkyne 38 as a colorless oil (33.1 mg,
71%). [a]2D0 =+46.2( c 1.66, CH2Cl2); 1H NMR (300 MHz, CDCl3): d=
7.20 (d, J=8.3 Hz, 2H), 6.86 (d, J=8.7 Hz, 2H), 6.27–6.15 (m, 1H), 5.81
(ddd, J=9.0, 6.1, 1.1 Hz, 1H, H16), 3.79 (s,
1H, -OH), 3.47 (dd, J=11.7, 8.9 Hz, 1H,
H17a), 3.39 (dd, J=11.7, 6.0 Hz, 1H, H17b),
2.90 (ddd, J=12.7, 8.5, 8.0 Hz, 1H, H4a), 2.59
(dddd, J=12.6, 7.3, 5.1, 1.0 Hz, 1H, H4b), 2.45
(m, 1H, H8), 2.33–2.37 (m, 2H, H5), 2.30
(ddt, J=14.2, 3.1, 1.8 Hz, 1H, H12a), 2.10
(ddd, J=14.7, 2.8, 2.0 Hz, 1H, H14a), 1.95
(dd, J=14.7, 3.5 Hz, 1H, H14b), 1.87 (d, J=
1.4 Hz, H19, 3H), 1.44–1.67 (m, 4H, H9,
Chem. Eur. J. 2007, 13, 135 – 149
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147

A. Fürstner et al.
H10), 1.37 (ddd, J=14.3, 11.7, 2.6 Hz, 1H, H12b), 1.14 (d, J=7.0 Hz, 3H,
H20); 13C NMR (150 MHz, CDCl3): d=174.7 (s, C18), 165.3 (s, C1),
156.1 (s, C3), 118.2(d, C2), 97.7 (s, C15), 86.3 (s, C7), 79.6 (s, C6), 68.8
(d, C13), 63.9 (d, C11), 61.6 (d, C16), 34.1 (t, C10), 33.4 (t, C12), 32.7 (t,
C4), 31.4 (t, C14), 31.3 (t, C9), 28.8 (t, C17), 25.6 (d, C8), 24.2 (q, C19),
22.7 (q, C20), 18.3 (t, C5).
Acknowledgements
Generous financial support by the MPG, the Chemical Genomics Center
(CGC Initiative of the MPG), the Fonds der Chemischen Industrie, the
Alexander-von-Humboldt Foundation (fellowships for M.D.B.F. and
C.N.), and the Deutsch-Israelische Projektkooperation (DIP project) is
gratefully acknowledged. We thank Dr. L. Parra-Rapado for the prepara-
tion of the bare macrocycle 52, Dr. C. W. Lehmann for the crystal struc-
ture analysis of compound 33, and Mrs. A. Langerak for excellent techni-
cal assistance.
Compound 50: Prepared as described above from ester 54 (12mg,
0.022 mmol)[13] and CAN (30 mg, 0.054 mmol); colorless oil (4.5 mg,
49%). [a]2D0 =+11.5 (c 0.41, CH2Cl2); 1H NMR (400 MHz, CD2Cl2): d=
6.42(dt, J=11.4, 7.2Hz, 1H), 5.86 (dt, J=11.8, 1.8 Hz, 1H), 5.73–5.68
(m, NH), 5.41 (quint., J=3.0 Hz, 1H), 4.10–4.02(m, 1H, OH), 3.81 (ddd,
J=8.8, 7.2, 1.0 Hz, 1H), 3.48 (dd, J=11.6, 8.8 Hz, 1H), 3.39 (dd, J=11.7,
6.3 Hz, 1H), 2.82 (qd, J=7.2, 1.8 Hz, 2H), 2.31–2.24 (m, 2H), 2.17–2.10
(m, 2H), 1.96 (d, J=3.2Hz, 2H), 1.89 (dt, J=14.4, 2.4 Hz, 1H), 1.76 (t,
J=2.7 Hz, 3H), 1.75 (t, J=2.7 Hz, 3H), 1.65–1.45 (m, 5H); 13C NMR
(75 MHz, CD2Cl2): d=174.4, 164.6, 150.8, 120.0, 97.6, 79.1, 78.0, 76.8,
76.0, 68.0, 65.3, 61.8, 35.0, 34.6, 31.8, 29.2, 29.0, 25.2, 18.9, 18.6, 3.5 (2C);
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MS (EI): m/z (%): 419 (8), 317 (3), 179 (52), 161 (19), 133 (34), 121 (33),
111 (46), 102(36), 93 (100), 77 (37); HRMS (ESI): m/z: calcd for
C22H29NO5S+Na: 442.16587; found: 442.16582 [M ++Na].
Compound 51:
A solution of PPh3 (16.5 mg, 0.063 mmol), DEAD
(8.4 mL, 0.063 mmol), alcohol 17 (20 mg, 0.048 mmol)[13] and heptanoic
acid (7.5 mL, 0.053 mmol) in benzene (2mL) was stirred for 24 h before
additional PPh3 (50 mg, 0.15 mmol), DEAD (25 mL, 0.15 mmol) and hep-
tanoic acid (23 mL, 0.15 mmol) were added. After 72h reaction time, the
solvent was evaporated and the residue was purified by flash chromatog-
raphy (hexanes/EtOAc 2:1 ! 1:1 ! 1:2) to give ester 55 which was used
in the next step without further characterization. CAN (40 mg,
0.074 mmol) was added to a solution of this ester (8 mg) in MeCN
(0.45 mL) and water (0.22 mL) and the resulting mixture was stirred for
20 h at ambient temperature. The reaction mixture was partitioned be-
tween CH2Cl2 and water and the organic layer was dried over Na2SO4
and concentrated. Purification of the residue by flash chromatography
(hexanes/EtOAc 2:1 ! 1:1) gave product 51 as a colorless oil (2.8 mg,
14%). [a]2D0 =+30 (c 0.25, CH2Cl2); 1H NMR (400 MHz, C6D6): d=5.72
(s, NH), 5.07–5.03 (m, 1H), 4.16 (dd, J=9.1, 4.1 Hz, 1H), 4.05–3.97 (m,
1H), 3.92(s, 1H), 3.79 (t, J=9.0 Hz, 1H), 3.22 (dd, J=9.0, 4.2Hz, 1H),
2.43–2.35 (m, 1H), 1.95 (td, J=7.6, 3.5 Hz, 2H), 1.74–1.67 (m, 1H), 1.64–
1.57 (m, 2H), 1.56 (d, J=2.4 Hz, 3H), 1.54–1.33 (m, 4H), 1.24–1.19 (m,
4H), 1.17 (d, J=6.8 Hz, 3H), 1.15–1.09 (m, 4H), 1.04–0.98 (m, 1H), 0.86
(t, J=7.2Hz, 3H); 13C NMR (75 MHz, C6D6): d=171.4, 159.5, 96.7, 83.7,
76.4, 68.0, 65.2, 64.7, 59.4, 34.5, 34.4, 33.7, 33.2, 32.1, 31.7, 29.0, 26.3, 25.2,
22.9, 21.9, 14.2, 3.5; IR (film): n˜ = 3348, 2921, 2858, 1740, 1407, 1241,
1170, 1097, 929, 803; MS (EI): m/z (%): 409 (5), 323 (3), 193 (89), 175
(27), 147 (46), 133 (75), 125 (60), 113 (42), 110 (11), 87 (34), 73 (52), 43
(100); HRMS (ESI): m/z: calcd for C22H35NO6:+Na: 432.23566; found:
432.23584 [M ++Na].
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identifies this residue as a glutamic acid moiety.
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X-Ray crystallographic study for compound 33: Data were recorded
using an Bruker-AXS KappaCCD-diffractometer with graphite-mono-
chromated MoKa radiation (l=0.71073 Š). The crystal was mounted in a
stream of cold nitrogen gas. The structures were solved by direct methods
(SHELXS-97)[46] and refined by full-matrix least-squares techniques
against F2 (SHELXL-97).[47] Hydrogen atoms were inserted from geome-
try consideration using the HFIX option of the program. Selected data:
C28H43NO6Si, Mr =517.72gmol ꢁ1
0.04 mm, orthorhombic, P212121 [No. 19], a=7.2255(3), b=12.6489(5),
m=
, colorless, crystal size 0.08”0.06”
c=32.6244(13) Š, V=2981.7(2) Š3, Z=4, 1calcd =1.153 Mgmꢁ3
,
0.117 mmꢁ1, T=100 K, 38101 reflections collected, 8662independent re-
flections, 5823 reflections with I>2s(I), qmax =30.018, 333 refined param-
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eters, R=0.067, wR2 =0.146, S=1.028, largest diff. peak and hole=0.6/
ꢁ3
ꢁ0.4 eŠ
.
CCDC-601356 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif/
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148
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Chem. Eur. J. 2007, 13, 135 – 149

Latrunculin Analogues
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Received: September 19, 2006
Published online: November 8, 2006
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149