Dictyostatin
A DaMocles project by:
Max Dombrowsky, Rico Ballmann, Valeska Botschek, Hanna Frühauf and Sebastian Barthel
Dictyostatin is a secondary metabolite of several sponges and a potential anticancer agent. Because
of its bad sourcing, it took many years until its great cytostatic character was established and some
total synthesis methods were developed. Currently the development of Dictyostatin is still in
progress and the medical usage is not available yet.
1. Chronological survey of Dictyostatin:
In the early 1970s scientists realised the
toxicity of secondary metabolites of several
naval organisms, which are used by these
organisms for defending themselves against
microalgae, bacteria and parasites growing on
them. Secondary metabolites are bioactive
non-essential compounds produced by the
organism itself or some microorganisms
associated in symbiosis with the organism.
The lethal effect of many secondary
metabolites, suggest the assumption that
many of these compounds might work as
cytostatic agents. Because of these
assumption and many proving investigations
naval secondary metabolites became
important targets in the anticancer research.
One of the first investigations, providing some
indications for the effect of secondary
metabolites, was realised by analysing the
hunting behaviour of the nautilus „Conus
magnus“. Conus magnus is using poisoned
arrows for paralysing prey. The used poison is
part of a group called „Conotoxin“, build out
of 10-30 amino acids, which are building up a
bioactive compound interrupting signal
transduction in neurons by blocking ion
channels. By using this knowledge, the first
distributed marine active agent „Ziconotide
(Prialt®)“ was developed.
It took a few years until other marine species
like sponges and corals became targets in
marine agent research. In 1993 Bob Pettit et al
from Arizona State University isolated a group
of anticancer agents called “Spongistatins” out
of Spongia sp. (Order: Dictyoceratida), a
sponge found near the Maldives. One year
later, in 1994, they isolated another secondary
metabolite out of Spongia sp. . They called this
compound “Dictyostatin”, due to a bad yield
of 1,35mg out of 400kg wet sponge, just a few
investigations were realised. But a tendency
was clearly recognisable. Dictyostatin showed
strong characteristics of a good cytostatic
agent. But it took nearly a decade until
another profitable source of Dictyostatin was
found. In 2003 Wright et al isolated the
compound in a better yield (of 2,8*10-3%) out
of a Carribean lithistid sponge (family:
Corallistidae), which they found by using a
manned submersible at great depth near
Jamaica. 1After some purification and
characterisation, they filed a patent
application and are currently the patent
holder of the method of using Dictyostatin for
inhibiting growth of tumour cells.2 There
whole investigations were realised in
cooperation with Harbor Branch
Oceanographic Institution (Florida Atlantic
University) and showed high impacts by low
concentrations of the agent. The inhibiting
growth concentration for inhibiting 50% of the
cells lies between 50pM and 1nM.3 In
addition, they recognised that Dictyostatin is
also active against some multidrug resistant
cells, which made Dictyostatin attractive as an
alternative to Taxol. The bad sourcing of high
quantities induced many scientists to work on
total synthesis of Dictyostatin. One of these
1
Stereochemical determination of dictyostatin
Paterson, I.; Britton, R.; Delgado, O.; Wright, A. E. Chem.
Commun. 2004, 6, 632-633
2
http://www.google.com/patents/US6576658,
07.07.2013 16:05
3
http://www.chem.wisc.edu/~burke/dictyostatin.htm,
07.07.2013 16:05
scientists is Ian Paterson, working in coop.
with Harbor Branch Oceanographic Institution,
who developed a method for a stereo
controlled total synthesis and investigated
different diastereomers of Dictyostatin.
2. Mode of operation of Dictyostatin
Dictyostatin is a 22-membered Macrolacton of
the familiy of Spongistatines. It is a cytotoxin
that leads to a cellcycle-arrest between the
G2- and the M-Phase and is active even in
nanomolar concentrations (GI50 between 0.69
nm and 1.49 nm on HeLa-cells).4 The cytoxicity
is made through stabilization of microtubules
or induction of the polymerisation of the
protofilaments.5 If the dynamic instability of
the filaments is damaged, they are not able to
Dictyostatin6
build up the spindle apparatus or
depolymerise to release cell-cell-contacts. As
this is necessary to entrance mitosis, the cell
will not pass the cell-cycle-checkpoint
between G2 – and the M-Phase. Mitosis and
cell-division is not possible in fact Apoptosis is
initiated.
Microtubules are a central target in cancer
research and drug discovery because they play
such a crucial role in cell division. They form
the spindle apparatus so that cells can
undergo mitosis and depolymerise to release
cell-cell contacts for cells are able to round up.
Microtubules itself are polymers that consist
4 7
Madiraju et al, Biochemistry, 2005, 44, 15053 - 15063
5 8
H. Röhm, Studie zur enantioselektiven Synthese des
marinen Makrolactons (-)-Dictyostatin, 2010, 15 - 20
6 9
http://www.marinebiotech.org/images_large/dictyostat
in.gif 4.7.13, 19:58
of heterodimers (α- and β-Tubulin). A
cytotoxic drug can manipulate the polymer
either through avoiding or promoting
depolymerisation. Common cytotoxins that
lead to the depolymerisation of microtubuli
are Colchizin (toxin of meadow saffron) and
Vinca-alcaloids (Madagascar periwinkle).
Paclitaxel (Pacific yew) from the familiy of
taxanes and Dictyostatin (Dictyoceratida).
Both – Paclitaxel and Dictyostatin – bind to a
binding pocket on β-Tubulin even if they are
structurally totally different. 7
The binding pocket is an emargination on β-
Tubulin next to the lateral contact between α-
and β-Tubulin.8 The cytotoxins are thus able to
stabilize the GDP-bond form of β-Tubulin that
is responsive for depolymerisation normally.
The difference between Dictyostatin and the
Taxanes lies in a mechanism that is called
Multidrug resistance (MDR). While Paclitaxel
shows no activity in cells, which express the
MDR-1 gen, Dictyostatin is effective even in
poly-resistant tumor cell lines.9
Multi-drug resistant cell lines use ABC-
transporters (ATP-binding cassettes) like p-
Glycoproteins (PGP) to export lipophile,
neutral or basic molecules under ATP-effort
from the cell. Therefore PGP build a canal
through the cell membrane and potential
cytotoxic substances can be excreted.
Molecules in the range of 400 to 2000 Dalton
can pass this canal so that especially tumour
cells can defend themselves from cytotoxic
drugs.
Another mechanism of cells’ self-defending is
the structural change of β-Tubulin to βIII-
Tubulin. The difference between the two
types of tubulin is a Phe270 in the binding
pocket. It is exchanged by a Valin so the
Soulére; ChemMedChem 2009, 4, 161 - 163
H. Röhm, Studie zur enantioselektiven Synthese des
marinen Makrolactons (-)-Dictyostatin, 2010, 15 - 20
H. Röhm, Studie zur enantioselektiven Synthese des
marinen Makrolactons (-)-Dictyostatin, 2010, 15 - 20
stabilizing effect of Taxanes gets lost because
of missing interaction between molecule and
binding pocket. The tested cell lines in this
case are 1A9PTX10 and 1A9PTX22 (human
ovarian adenocarcinoma).10
The reason why this mechanism exists is a
simple one. The cells defend themselves
against the accumulation of toxic substances
especially in brain cells. In case of the brain
this is called the haemato-encephalic barrier
and therefore cerebral tumours are so hard to
medicate.11
In matters of Dictyostatin this mechanism
seems to show no effect so that it attracted
notice of the researchers. The main reason for
this phenomenon is the ability to bind to βIII-
Tubulin as well, even if there are structural
changes. To get an effect on βIII-Tubulin cells
(1A9PTX10) with Paclitaxel one needs 90fold
concentration, whereas Dictyostatin is active
in only 4.6fold concentration. The greater
affinity to the binding-pocket at all is shown in
the fact that Dictyostatin is able to polymerize
microtubules without the presence of GTP or
MAPs (microtubule associated proteins)
(Picture A: complete system with MAPs and
GTP, Picture B: only Tubulin).12 This seems to
be obvious referring to the capacity of
Dictyostatin to stabilize GDP-bound Tubulin.
Paclitaxel13
The advantages in comparison to Paclitaxel
are seen when both cytotoxins are present in
the system. Then Dictyostatin will displace
10
Madiraju et al, Biochemistry, 2005, 44, 15053 - 15063
11
http://www.pharmazeutische-
zeitung.de/index.php?id=2381, 4.7.13, 17:52
12
Madiraju et al, Biochemistry, 2005, 44, 15053 - 15063
13
http://upload.wikimedia.org/wikipedia/commons/thu
mb/5/59/Taxol.svg/250px-Taxol.svg.png 4.7.13, 20:00
Paclitaxel from the binding-pocket.14 Probably
it is the affinity that makes Dictyostatin
resistant against the ABC-Transporters as
well.15
The remarkable question now is the reason for
these special abilities. A part of the answer is
found in the research on interdependency of
Dictyostatin-derivates with the binding-
pocket.
The region of C14 and C16 and the C2-C3 –
double bound seem to be of prime
importance. If the C16 – stereocenter is
inverted (R instead of S in 16-epi-dictyostatin)
there are awkward interactions with the
phe270 in the “normal” binding-pocket of β-
Tubulin (tested on HeLa-cells) and now
cytotoxic effect can be observed. In
comparison the 16-Normethyldictyostatin
(loss of methylgroup on C16) is still active but
3.5fold less.
In the test with the β-Tubulin mutants (βIII-
Tubulin, 1A9PTX10 with Valin) the C16-
methygroup seems to be crucial. The
methylgroup is responsive for interactions
with the valin in the binding-pocket and
without this interaction no binding seems to
be possible. This argumentation can be
transferred to the methylgroup on C14.
Further requirements are the Z-configuration
of the C2-C3-double bound whereas
unsaturated C-atoms are not mandatory. The
effect of saturated bounds is nearly the same.
The S-configuration of C9 is required as well.
The accuracy of the fit between Dictyostatin
and binding-pocket is seen in the result that
the open-chained methylester (not as a
macrolacton) shows activity as well because it
has the same 3D-configuration. In comparison
the so called iso-Dictyostatin which is an 20-
membered macrolacton shows obviously
cytotoxic effect because its 3D-structure is
completely different.11
14
Madiraju et al, Biochemistry, 2005, 44, 15053 - 15063
15
H. Röhm, Studie zur enantioselektiven Synthese des
marinen Makrolactons (-)-Dictyostatin, 2010, 15 - 20
A: complete system with MAPs and GTP B: only tubulin

3. Molecules with similar characteristics as of microtubules and promoting their

Dictyostatin depolymerisation.

Dictyostatin is not the only compound
inhibiting cell division by stabilising
microtubules. Many cytostatic agents use the
same mechanism with slide differences, but
even totally different ways are possible.
There are many different examples who to
inhibit cell division. One example are some
antimetabolites, which are displacing
metabolites of the active site of an enzyme
and thereby inhibiting the enzyme
competitively. Some other antimetabolites are
imitating bases of the DNA or they are
alkylating nucleic acids. Both ways are
damaging the DNA with the purpose of
preventing any DNA replication.
Dictyostain likely agents are, beside of the
Paclitaxel, Docetaxel another member of the
group of the taxanes like Paclitaxel. Taxanes
have a totally different structure than
dictyostatin and structural likely compounds
like Epothilone, Peloruside & Discodermolide.
Epothilone and Discodermolide are using the
same mechanism like Dictyostatin, but
unfortunately both agents have dangerous
side effects, like nerve damages caused by
Epothilones and lungs toxicity caused by
Discodermolides. Currently they are not
accessible for practical application, but drug
research is trying to restrain their side effects.

Another group of agents is acting on

microtubules, like Paclitaxel and Dictyostatin.
The most common way for this group is
promoting or inhibiting microtubule-
(de)polymerisation. While agents like
dictyostatin are stabilising microtubules and
promoting their polymerisation, some others
Epothilon A16
use the second ways of destabilising. One
example for this subgroup are vinca alkaloids.
These agents are binding next to the GTP
binding site of tubulin, inhibiting the dynamic

16
http://upload.wikimedia.org/wikipedia/commons/b/b
5/Epothilon_A.png 07.07.2013 17:17
Discodermolide17 Dictyostatin18

Docetaxel19 Paclitaxel20

4. Total synthesis of (-)-Dictyostatin21

We have seen so far that (-)-Dictyostatin is a molecule which has a huge amount of stereogenic
centers: There are 4 stereo chemical C-C double bonds and about 11 stereogenic centers within the
molecule, influencing its activity. Because of these circumstances a total synthesis is difficult and very
likely to produce a lot of waste. Another problem is the unknown path of natural synthesis, which
leads us to the fact, that a biotechnical method Dictyostatin’s production is still a dream of the
future. Despite all of these issues, there are some paths of total synthesis which have yield of up to
3%.
The most effective path was discovered by Ian Paterson et al., with an effectiveness of about 3.8%.
His synthesis is influenced by the already known synthesis of (+)-Discodermolid which has a very
similar structure and stereo chemical configuration.

17
http://en.wikipedia.org/wiki/File:Discodermolide_Structure.png 07.07.2013 17:20
18
http://www.marinebiotech.org/images_large/dictyostatin.gif 4.7.13, 19:58
19
http://commons.wikimedia.org/wiki/File:Docetaxel.svg 07.07.2013 17:21
20
http://upload.wikimedia.org/wikipedia/commons/thumb/5/59/Taxol.svg/250px-Taxol.svg.png 4.7.13 20:00
21
Hartmut Röhm, Studien zur enantioselektiven Synthese des marinen Makrolactons (-)-Dictyostatin (Dissertation der
Fakultät für Chemie und Pharmazie der Berhard Karls Universität Tübingen)
First of all Paterson’s idea was to synthesise the double bond found at C10 by a Still-Gennari modified
Horner-Wadsworth-Emmons-reaction, leading to the C11-C26 fragment (2-21) and the C1-C10
fragment which could be synthesised by using Stille reaction to make the bond between C3 and C4,
leading to 2-26 and 2-27.

The C11-C26 fragment can be synthesized by using Horner-Wadsworth-Emmons- reaction again,

leading to the aldehyde 2-22 and the ketophosphonate 2-23.

As you can see, the absolute configuration between the C12-C14, within 2-22, and C20-C22, within 2-
23, is equal and can be related to the same precursor 2-24 which can be synthesized from (S)-Roche
ester (methyl- (S)-3-hydroxy-2-methylpropionate).

To do so the (S)-Roche ester was transposed with p-Methoxybenzyltrichloracetimidate under acid

conditions to deploy the PMB group. Then Weinreb-Nahm ketone synthesis by using
Ethylmagnesium bromide, Paterson aldol reaction by using formaldehyde and reduction by using
Natriumtriacetoxyborhydrid leads to the precursor 2-24 with an effectiveness of about 52%, because
of the nonstereoselective last synthesis step.

Precursor 2-24 was used to synthesize aldehyde 2-22. First both hydroxylgroups were silylated, then
the hydroxyl group at C15 was solvolysed to alcohol which was transposed to iodide under modified
Mitsunobu conditions by using PPh3, Iodide and Imidazole. Secound step was a Myers-reaction with
N-propionylised pseudoephedrin derivate 2-35 leading to 2-36 which was reduced by LDA and
BH3NH3 and subsequently oxidized by DMP and NaHCO3 to the aldehyde 2-22 with an effectiveness
of about 55%.

To produce ketophosphonate 2-23 Paterson used the precursor 2-24, silylated the hydroxyl group at
C19, transposed it with DDQ(2,3-Dichlor-5,6-dicyan-1,4-bzochinon), opend it with

DIBAL-H and used Dess-Martin oxidation to produce the aldehyde 2-40.

Nozaki-Hiyama-Kishi reaction with Bromallyltrimethylsilane in CrCl2 deployed the terminal dien 2-42
which was desilylated by camphorsulfonic acid and oxidized by Dess-Martin periodinane. The
resuming aldehyde was now phosphonated by (MeO)2P(o)CH2Li and oxidized to ketophosphonate 2-
23 with an effectiveness of about 48%.

Horner-Wadswoth-Emmons-reaction with aldehyde 2-22 and phosphonate 2-23 led to ketoenon 2-

45 which was reduced by Stryker’s reagent, oxidatively deprotected by DDQ and syn-selectively
reduced by Zn(BH4)2, leading to 2-46. After that C11 and C19 hydroxyl groups were protected, while
the C21 hydroxyl group wasn’t protected due to steric effects. Subsequently C11 hydroxyl group was
deprotected by Tertbutylammoniumflouride and oxidized by 2,2,6,6-Tetramethyl-1-piperidinyloxide
(TEMPO), leading to the C11-C26 fragment 2-21 with an effectiveness of about 52%.
Fragment C4-C10 was made by using aldehyde 2-49. The C6,C7 anti configuration was deployed by
using Brown crotylboration. Afterwards the now existing C7 hydroxyl group was silylated, the double
bond in C4,C5 was ozonolysated and methylated using the Takai-reaction. The C9 hydroxyl group was
now desilylated and oxidized by DMP and Sodium chlorite resulting in acid 2-53. Subsequently the
acid was chlorinated by Ghosez’ reagent 2-54 and finally Lithiumphosphonate 2-55 was added,
leading to the C4-C10 fragment 2-26 with an effectiveness of about 24%

Finally the C11-C26 fragment 2-21 and the C4-C10 fragment 2-26 is coupled by using the Still-Gennari
modified Horner-Wadsworth-Emmons-reaction, which uses 18-crown-6 and K2CO3, and is in this
case (z)-selective (Z/E=5:1). 2-27 was added at C4 by using Liebeskind modified Stille-reaction, using
CuTC (TC=Thiophen-2-carboxylate) and potassium fluoride, leading to the Dictyostatin base frame 2-
58.

The C1 triisopropylsilyl-ester (CO2TIPS) was now saponificated and afterwards the lactone was
deployed under Yamaguchi conditions. Subsequently the C9 Ketone was reduced under Luche
conditions and finally C7 and C19 hydroxyl groups were deprotected with hydrochloric acid in MeOH.

The effectiveness of the last cascade is about 29% leading to a global effectiveness of approximately
3.8%
Aside from Paterson’s path there are some other ways to synthesise Dictyostatin:

 Curran et al. global effectiveness: ~1%

 Philips et al. global effectiveness: ~1%
 Ramachandran global effectiveness: ~3%

There are also some other ways to produce parts of the molecule with the same stereochemical
configuration, but the most effective path is currently the one of Ian Paterson.