New synthetic strategies towards psammaplin A, access to natural product analogues for biological evaluation

Article abstract of DOI:10.1039/c0ob00824a

New synthetic routes towards the natural product psammaplin A were developed with the particular view to preparing diverse analogues for biological assessment. These routes utilize cheap and commercially available starting materials, and allowed access to

Full text of DOI:10.1039/c0ob00824a

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 659
www.rsc.org/obc
COMMUNICATION
New synthetic strategies towards psammaplin A, access to natural product
analogues for biological evaluation†
Matthias G. J. Baud,a Thomas Leiser,b Franz-Josef Meyer-Almesb and Matthew J. Fuchter*a
Received 4th October 2010, Accepted 12th November 2010
DOI: 10.1039/c0ob00824a
New synthetic routes towards the natural product
psammaplin A were developed with the particular view
to preparing diverse analogues for biological assessment.
These routes utilize cheap and commercially available starting
materials, and allowed access to psammaplin A analogues
not accessible via currently reported methods. Preliminary
biological studies revealed these compounds to be the most
potent non peptidic inhibitors of the enzyme histone deacety-
lase 1 (HDAC1, class I) discovered so far. Interestingly,
psammaplin A and our synthetic analogues show class I
selectivity in vitro, an important feature for the design and
synthesis of future isoform selective inhibitors.
regulation of gene expression, and misregulation of their activity
has been found to be involved in cancer pathogenesis.8a–c HDAC
and DNMT enzymes therefore represent promising targets for the
development of anticancer therapies. Indeed, there is increasing
interest in epigenetic therapies, in part due to the success of DNMT
and HDAC inhibitors, such as decitabine and vorinostat, in the
clinic and their recent FDA approval for use in certain tumour
types. In vitro, psammaplin A (and several other psammaplins),
displayed potent activity against an HDAC cell extract (IC50
=
4.2 nM) and DNMT1 (IC50 = 18.6 nM). Subsequently, studies on
its anti-proliferative properties have shown it to have significant
cytotoxicity (ED50, mg mL-1) against human lung (A549, 0.57),
ovarian (SK-OV-3, 0.14), skin (SK-MEL-2, 0.13), CNS (XF498,
0.57), and colon (HCT15, 0.68) cancer cell lines.9 In vivo, it
inhibited tumour growth in the A549 lung xenograph mouse model
while maintaining low toxicity.1a
Psammaplin A (1, Fig. 1), is a member of a family of natural
products isolated from several marine sponges including Pseudo-
ceratina purpurea.1a
Our laboratory is currently involved in several medicinal chem-
istry projects, focusing on the modulation of enzymes involved
in the epigenetic regulation of gene expression. We considered
that access to diverse psammaplin A analogues would enable
the establishment of structure–activity relationships (SARs) to
explore its reported potency against HDAC and DNMT enzymes.
To date, several total syntheses of psammaplin A have been
reported10–12 (Scheme 1), starting from tyrosine or phenylpyruvic
acid derivatives, with minimal analogues reported. These syntheses
suffer from the low commercial availability of tyrosine and
phenypyruvic acid derivatives, notably with diverse aromatic
substitution patterns. We therefore considered the development of
alternative synthetic routes with improved substrate scope to allow
the synthesis of biologically interesting analogues. In general,
bromotyrosine derivatives represent a diverse class of marine
natural products structurally related to psammaplin A (Fig. 2)
and such synthetic procedures should facilitate their preparation
and further study.13,14
To overcome the shortcomings of prior routes we considered
the well known 2 step Erlenmeyer oxazolone synthesis–hydrolysis
sequence as a viable route to a variety of arylpyruvic acids,
which utilizes cheap and commercially available substituted ben-
zaldehydes 11 as substrates (Scheme 2). Upon exposure to N-
acetyl glycine and acetic anhydride in the presence of sodium
acetate, aldehydes 11 were converted to oxazolones 12. Further
treatment with aqueous HCl afforded arylpyruvic acids 13. The
structurally diverse acids 13 generated were used as precursors
to psammaplin A analogues following the previously reported
Fig. 1 Psammaplin A (1).
Numerous bromotyrosine derivatives have been isolated from
these marine sponges,1a–h notably from sponges of the order
Verongida, known to be a rich source of such metabolites.2
Psammaplin A was structurally characterized in 1987,1b–d and
represents the first example of a disulfide-containing metabolite
isolated from a marine sponge. While it has been implicated as
an inhibitor of numerous targets such as topoisomerase II,3 DNA
gyrase,4 leucine aminopeptidase,1g farnesyl protein transferase,1g
chitinase,1f mycothiol-S-conjugate amidase,5 aminopeptidase N6
and DNA polymerase a-primase,7 studies by Crews and co-
workers showed it to be an extremely potent enzymatic inhibitor of
both histone deacetylases (HDACs) and DNA-methyltransferases
(DNMTs).1a These enzymes play a crucial role in the epigenetic
aImperial College London, South Kensington Campus, Department of
Chemistry, London, UK SW7 2AZ. E-mail: m.fuchter@imperial.ac.uk
bUniversity of Applied Sciences, Department of Chemical Engineering and
Biotechnology, Schnittspahnstrabe 12, 64287 Darmstadt, Germany
† Electronic supplementary information (ESI) available: Experimental
procedures, biological assay procedures and full characterization for all
new compounds. See DOI: 10.1039/c0ob00824a
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 659–662 | 659
©

View Article Online
Fig. 2 Examples of bromotyrosine based natural products.
Scheme 1 Reported syntheses of psammaplin A.
routes.10–12 This sequence allowed us to synthesise psammaplin
A and a collection of more than 70 psammaplin A analogues.
Representative examples are given in Scheme 2.
We faced several synthetic issues however when starting
with electron rich benzaldehydes, such as p-dimethylamino-
benzaldehyde. While the Erlenmeyer oxazolone synthesis is effi-
cient in the case of electron poor aromatic aldehydes, low yields
are generally obtained for electron rich substrates. Moreover,
adjustment of the pH and isolation of the subsequent arylpyruvic
acids can become difficult when the aromatic ring bears basic (e.g.
amino) functionality. Finally, the strong acidic work-ups involved
in the condensation and coupling steps of reported syntheses make
these routes unsuitable in the case of compounds containing basic
and/or acid-sensitive functionality.
To overcome these problems, we developed alternative routes to
psammaplin A and analogues non-accessible via this procedure.
Our retrosynthetic analysis is given in Scheme 3. Product 16 would
be accessible by double amidation of ester 17 and condensation
with hydroxylamine to introduce the oxime unit. Ester 17 would
be accessible from unsaturated ester 18, via dihydroxylation and
regioselective dehydration. Unsaturated ester 18 would be ob-
tained from either Knoevenagel–Doebner condensation between
aromatic aldehyde 11 and 3-ethoxy-3-oxopropanoic acid 19, or
Scheme 2 Erlenmeyer oxazolone synthesis–hydrolysis path.
Heck coupling between aromatic halide 20 and methyl acrylate 21.
Such substrates are commercially available and cheap reagents.
Knoevenagel–Doebner condensation between 22 and 3-ethoxy-
3-oxopropanoic acid 19 afforded unsaturated ester 23 in excellent
yield (Scheme 4). The latter was converted in high yield to
diol 24 with osmium tetroxide in a water–acetonitrile mixture.
Regioselective dehydration of diol 24 with catalytic amounts of
p-TsOH in refluxing benzene, followed by condensation with
hydroxylamine afforded ester 25 in good yield after 2 steps, as
a single isomer. Compound 25 has been previously reported
in the literature during the synthesis of the natural product
verongamine by Spilling et al.,15 and its structure confirmed
by X-ray crystallography.16 Comparison of our data with that
reported matched perfectly. This protecting-group free sequence
employs mild reaction conditions, and represents a considerable
advantage compared to the use Nakamura’s a-OTBS-protected
660 | Org. Biomol. Chem., 2011, 9, 659–662
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Scheme 5 Synthesis of 32 and 33.
Scheme 3 Retrosynthetic analysis.
dehydration was thought to be particularly efficient in the case
of electron-rich aromatics, due to increased stabilization of the
positively charged benzylic position involved in the elimination
process. Amidation as before afforded 32 in good yield.
Analogue 33 was obtained following a similar sequence
(Scheme 5). Bromination of diol 28, followed by dehydration–
condensation and final amidation afforded 33 in moderate yield.
The reaction conditions for the syntheses of 1, 32 and 33 were
not significantly optimized, suggesting that higher yields are
potentially achievable, notably for the dehydration and coupling
steps.
In light of their potent reported activity against Class I HDACs,
psammaplin A and our synthetic analogues were evaluated in
in vitro assays against HDAC1 and HDAC6. Psammaplin A is
thought to act as a prodrug, inhibiting HDAC activity following
intracellular reduction of the disulfide moiety, generating the
corresponding thiol.19 Indeed, in our assay, the reduced form
was found to be more potent in each case. IC50 values for
reduced forms are given in Table 1. Both psammaplin A and
our synthetic analogues were found to be extremely potent and
selective against HDAC1, with IC50 values ranging from low nM
to pM in their reduced form, therefore more potent than reference
compounds trichostatin A20a–c or the FDA approved compound
SAHA (vorinostat).21a–d Moreover, comparison of HDAC1 (class
I) and HDAC6 (class II) data showed an interesting selectivity
for class I HDACs over class II. Further studies are underway
in order to understand the observed selectivity towards HDAC1
and HDAC6. Interestingly, methylthioether 15c was found to be
completely inactive, which is in full agreement with the thiol
hypothesis for the active species. Full biological assessment of
our library against both HDAC and DNMT enzymes is underway
and will be the subject of a future manuscript.
Scheme 4 Newly developed synthetic route.
dimethylphosphonate in a Horner–Wadsworth–Emmons reaction
with aromatic aldehydes.14,17,18
Heating ester 25 in the presence of 0.5 equivalents of cystamine
in methanol led to no reaction after extended periods of time,
despite the successful application of these conditions to the
synthesis of the natural product verongamine.15 Instead the best
results were obtained in the presence of trimethylaluminium,
however the choice of the solvent system, reaction time and
quantity of aluminium reagent were found to be crucial. Indeed,
relatively apolar organic solvents, such as CH2Cl2 and CHCl3,
led to no reaction or very low yields respectively. This was
principally attributed to low substrate solubility. Solvent polarity
was therefore varied and the use of a CH2Cl2–MeCN mixture
allowed the formation of psammaplin A in good yield.
This synthetic route was successfully employed for the syn-
thesis of analogues 32 and 33 (Scheme 5). Unsaturated ester
27 was prepared via a Heck reaction between 4-bromo-N,N-
dimethylbenzenamine 26 and methyl acrylate 21. Dihydroxylation
afforded diol 28 in good yield. We were pleased to observe
quantitative formation of ester 30 in 2 steps. The regioselective
In summary, we have developed several novel and expedient
routes towards psammaplin A and a variety of synthetic analogues
bearing electron rich or electron poor aromatics. The developed
strategies allow both aromatic aldehydes and aromatic halides
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 659–662 | 661
©

View Article Online
Table 1 HDAC1 assays, IC50 values (mM)
M. Legg, B. Synstad and D. M. F. van Aalten, Bioorg. Med. Chem.,
2002, 10, 1123; (g) J. Shin, H.-S. Lee, Y. Seo, J.-R. Rho, K. W. Cho and
V. J. P a u l , Tetrahedron, 2000, 56, 9071; (h) N. B. Pham, M. S. Butler
and R. J. Quinn, J. Nat. Prod., 2000, 63, 393.
2 P. R. Bergquist and R. J. Wells, Marine Natural Products, Academic
Press, New York, 1983, vol. 5.
3 D. Kim, I. S. Lee, J. H. Jung, C. O. Lee and S. U. Choi, Anticancer Res.,
1999, 19, 4085.
4 D. Kim, I. S. Lee, J. H. Jung and S. I. Yang, Arch. Pharm. Res., 1999,
22, 25.
a
cpd
R1
R2
R3
HDAC1
HDAC6
5 G. M. Nicholas, L. L. Eckman, S. Ray, R. O. Hughes, J. A. Pfefferkorn,
S. Barluenga, K. C. Nicolaou and C. A. Bewley, Bioorg. Med. Chem.
Lett., 2002, 12, 2487.
6 J. S. Shim, H. S. Lee, J. Shin and H. J. Kwon, Cancer Lett., 2004, 203,
163.
7 Y. Jiang, E. Y. Ahn, S. H. Ryu, D. K. Kim, J. S. Park, H. J. Yoon, S.
You, B. J. Lee, D. S. Lee and J. H. Jung, BMC Cancer, 2004, 4, 70.
8 (a) C. B. Yoo and P. A. Jones, Nat. Rev. Drug Discovery, 2006, 5, 37;
(b) R. W. Johnstone, Nat. Rev. Drug Discovery, 2002, 1, 287; (c) M.
Rodriguez, M. Aquino, I. Bruno, G. De Martino, M. Taddei and L.
Gomez-Paloma, Curr. Med. Chem., 2006, 13, 1119.
1
3-Br-4-OH
“
3-OMe-4-SMe
“
3-NO2
“
3-Br-4-OMe
4-NMe2
H
“
H
“
H
“
Me
H
“
dimer
H
dimer
H
dimer
H
Me
dimer
H
dimer
H
0.045
0.001
1.67
0.0006
0.50
0.001
>50
3.64
1.23
0.36
>50
1.42
7.75
1.21
>50
>50
2.33
>50
0.70
1R
15a
15aR
15b
15bR
15c
32
32R
33
“
0.001
0.18
0.004
3-Br-4-NMe2
“
H
“
33R
9 Y. Park, Y. Liu, J. Hong, C. O. Lee, H. Cho, D. K. Kim, K. S. Im and
J. H. Jung, J. Nat. Prod., 2003, 66, 1495.
10 O. Hoshino, M. Murakata and K. Yamada, Bioorg. Med. Chem. Lett.,
1992, 2, 1561.
a Thiols (XR) were obtained by in situ reduction of the corresponding
disulfides, using tris(2-carboxyethyl)phosphine hydrochloride (TCEP)
11 K. C. Nicolaou, R. Hugues, J. A. Pfefferkorn, S. Barluenga and A. J.
Roecker, Chem.–Eur. J., 2001, 7, 4280.
12 A. M. Godert, N. Angelino, A. Woloszynska-Read, S. R. Morey, S. R.
James, A. R. Karpf and J. R. Sufrin, Bioorg. Med. Chem. Lett., 2006,
16, 3330.
13 J. Peng, J. Li and M. T. Hamann, Alkaloids: Chem. Biol., 2005, 61, 59.
14 F. Hentschel and T. Lindel, Synthesis, 2010, 2, 181.
15 T. R. Boehlow, J. J. Harburn and C. D. Spilling, J. Org. Chem., 2001,
66, 3111.
to be used as substrates. Interestingly, preliminary biological
assays have shown our synthetic analogues to be extremely
potent HDAC inhibitors, more potent than current inhibitors
SAHA,21a–d trichostatin A20a–c or indeed psammaplin A itself.
Further biological studies are ongoing in order to understand
the observed selectivity towards HDAC1 over HDAC6.
16 N. P. Rath, T. R. Boehlow and C. D. Spilling, Acta Crystallogr., Sect.
C: Cryst. Struct. Commun., 1995, C51, 2654.
17 E. Nakamura, Tetrahedron Lett., 1981, 22, 663.
Acknowledgements
18 R. Plantier-Royon, D. Anker and J. Robert-Baudouy, J. Carbohydr.
Chem., 1991, 10, 239.
This work was supported by the Association for International
Cancer Research (AICR) (08-0407).
19 D. H. Kim, J. Shin and H. J. Kwon, Exp. Mol. Med., 2007, 39, 47.
20 (a) M. Yoshida, M. Kijima, T. Akita and T. Beppu, J. Biol. Chem., 1990,
265, 17174; (b) M. Yoshida, S. Horinouchi and T. Beppu, BioEssays,
1995, 17, 423; (c) L. Qiu, M. J. Kelso, C. Hansen, M. L. West, D. P.
Fairlie and P. G. Parsons, Br. J. Cancer, 1999, 80, 1252.
21 (a) V. M. Richon, Y. Webb, R. Merger, T. Sheppard, B. Jursic, L. Ngo,
F. Civoli, R. Breslow, R. A. Rifkind and P. A. Marks, Proc. Natl. Acad.
Sci. U. S. A., 1996, 93, 5705; (b) V. M. Richon, S. Emiliani, E. Verdin,
Y. Webb, R. Breslow, R. A. Rifkind and P. A. Marks, Proc. Natl. Acad.
Sci. U. S. A., 1998, 95, 3003; (c) L. A. Cohen, S. Amin, P. A. Marks,
R. A. Rifkind, D. Desail and V. M. Richon, Anticancer Res., 1999, 19,
4999; (d) B. S. Mann, J. R. Johnson, M. H. Cohen, R. Justice and R.
Pazdur, Oncologist, 2007, 12, 1247.
Notes and references
1 (a) I. C. Pina., J. T. Gautschi, G. Y. S. Wang, M. L. Sanders, F. J. Schmitz,
D. France, S. Cornell-Kennon, L. C. Sambucetti, S. W. Remiszewski,
L. B. Perez, K. W. Bair and P. Crews, J. Org. Chem., 2003, 68, 3866;
(b) E. Quinoa and P. Crews, Tetrahedron Lett., 1987, 28, 3229; (c) L.
Arabshahi and F. J. Schmitz, J. Org. Chem., 1987, 52, 3584; (d) A. D.
Rodriguez, R. K. Akee and P. J. Scheuer, Tetrahedron Lett., 1987, 28,
4989; (e) C. Jimenez and P. Crews, Tetrahedron, 1991, 47, 2097; (f) J. N.
Tabudravu, V. G. H. Eijsink, G. W. Gooday, M. Jaspars, D. Komander,
662 | Org. Biomol. Chem., 2011, 9, 659–662
This journal is
The Royal Society of Chemistry 2011
©