Synthesis of the putative structure of 7-deoxycylindrospermopsin: C7 oxygenation is not required for the inhibition of protein synthesis

Article abstract of DOI:10.1002/anie.200500520

(Chemical Equation Presented) Naturally occurring! The cyanobacterial metabolite 7-deoxycylindrospermopsin has been synthesized and its natural occurrence confirmed by HPLC. Structural analysis and protein-inhibition studies show that the uracil unit does

Full text of DOI:10.1002/anie.200500520

Angewandte
Chemie
Scheme 1. Structures of the cylindrospermopsins and chlorination
products.
Natural Product Synthesis
Synthesis of the Putative Structure of
7-Deoxycylindrospermopsin: C7 Oxygenation
Is Not Required for the Inhibition of Protein
Synthesis**
remains unknown. Investigations into the removal of these
toxins from water supplies have led to the isolation of both 5-
chlorocylindrospermopsin (3) and cylindrospermic acid (4).[4]
Both of these by-products were shown to be nontoxic by
mouse bioassay. Furthermore, a related metabolite (5),
described as 7-deoxycylindrospermopsin, has been isolated
and shown to be nontoxic.[5] Interestingly, 5 is thought to exist
as a mixture of tautomers (which are illustrated in Scheme 1),
as its 1H NMR spectrum lacked a signal for the vinylic proton
on the uracil unit and the resonances were generally broad.
These structure–activity relationships have led to conjecture
about the steric and electronic requirements of the uracil unit
and its role in the toxicity of these compounds.[1,4]
Our continuing interest in the synthesis of these intriguing
natural products and elucidation of their mode of action led
us to pursue the synthesis of 7-deoxycylindrospermopsin for
several reasons:[6] First, the absorption maximum (lmax) for 5
at 263 nm, which is consistent with the presence of a uracil
unit and not the tautomers shown in Scheme 1.[7] Second, the
configuration of C7 arises biosynthetically from the methyl
group of an acetate unit;[8] thus, the synthesis of 7-deoxy-
cylindrospermopsin would allow us to explore its intermedi-
acy in the biogenesis of 1 and 2. Last, the involvement of
cytP450 oxidases in the toxicity of 1 suggests a possible
oxidation event at C7,[9] thus potentially explaining the
redundancy of the configuration of C7 and also implicating
7-deoxycylindrospermopsin as a potential toxin if it inter-
cepted a common metabolic pathway.
Ryan E. Looper, Maria T. C. Runnegar, and
Robert M. Williams*
Cylindrospermopsin (1) and its naturally occurring epimer, 7-
epi-cylindrospermopsin (2; Scheme 1), are attracting increas-
ing attention as threats to public health.[1] These alkaloids are
hepatotoxic metabolites of the cyanobacterium Cylindrosper-
mopsis raciborskii and of three other types of cyanobacteria,
and they pose a serious public-health problem when they
occur in water supplies. Both 1 and 2 are potent inhibitors of
protein synthesis both in vitro (IC50 = 200 and 480 nm, respec-
tively) and in vivo (IC50 = 1.28 and 2.66 mm, respectively).[2]
number of researchers have shown that these compounds
inhibit the translation of mRNA into protein.[2,3] Despite two
decades of research, the exact mechanism of this inhibition
A
[*] R. E. Looper, Prof. Dr. R. M. Williams
Department of Chemistry
Colorado State University
Fort Collins, CO 80523 (USA)
Fax: (+1)970-49-1-3944
E-mail: rmw@chem.colostate.edu
Dr. M. T. C. Runnegar
Research Center for Liver Diseases
University of Southern California Medical Center
Los Angeles, CA 90033 (USA)
Noting that the optical rotation of 5 has not been
reported, we began by synthesizing it in a racemic fashion
(Scheme 2). Diimide-mediated coupling of 3-buten-2-ol with
N-Boc-glycine (6) afforded an allylic ester that underwent a
smooth enolate Claisen rearrangement on treatment with
2.2 equivalents of NaHMDS.[10] Reduction of the acid and
acidic removal of the Boc group gave crotyl glycinol salt 7 in
[**] This work was supported by the National Institutes of Health (NIH)
Grants GM068011 and DK51788 (to R.M.W. and M.T.C.R, respec-
tively) and the National Science Foundation Grant CHE0202827 (to
R.M.W). The Cell Culture Core of the USC Center for Liver Disease
(P30 DK 48522) provided the rat hepatocytes used in these studies.
We are grateful to Array Biopharma for fellowship support to R.E.L.
We thank Dr. A. Humpage of the Australian Water Quality Center,
South Australia for providing a sample of natural 7-deoxycylidro-
spermopsin; Dr. G. Shaw for providing a spectra of 5; and Dr. C.
Rithner for helpful discussions concerning the NMR analysis.
good yield. Isourea 10 was obtained in nine further steps.[6b]
A
highlight of this sequence is the intramolecular dipolar
cycloaddition to afford 8, in which three contiguous stereo-
genic centers were constructed[6a] in ring A in a selectivity of
approximately 10:1. An acid-mediated oxidation employing
tetramethylpiperidine-1-oxyl allowed a selective oxidation of
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200500520
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Communications
The reductive guanidinylation sequence is sufficiently
clean to effect the direct sulfonation of 14 and 15 at O12 to
give sulfates 16 and 17 in a combined yield of 66% from 13
after a single purification by HPLC. In the 1H NMR spectrum
of 16, the uracil proton at C5 is clearly evident at d =
5.72 ppm, and the resonances are well defined and closely
match those of 1, except for the methylene protons at C7. In
an attempt to reconcile these differences, this spectrum was
compared with the spectrum that had led to the elucidation of
the structure of 5. However, it is clear that the natural
material is a mixture of compounds, and it was not possible to
conclude whether 16 was a minor component of that mixture.
Most significantly, our synthetic 7-deoxycylindrospermop-
sin (16) proved to be a potent inhibitor of protein synthesis
in vitro, as measured in the rabbit reticulocyte lysate sys-
tem.[9b] Protein synthesis was completely inhibited at 12 mm
and partially inhibited at 500 nm. This effect on protein
synthesis was also evaluated in whole cells (Table 1).[2] As
seen, 16 completely inhibits protein synthesis at 10 mm, thus
displaying a potency that is within an order of magnitude of
natural 1. Synthetic 16 also inhibits the synthesis of gluta-
thione (GSH), in a similar fashion to 1.[3,9a]
Scheme 2. a) 3-Buten-2-ol, DIC, DMAP, CH2Cl2 (96%); b) 2.2 equiv
NaHMDS, THF, 08C!RT (99%); c) EtOCOCl, Et3N, THF, NaBH4,
H2O; d) AcCl, MeOH (60%, 2 steps); e) BrCH2CO2Ph, iPr2NEt, MeCN
(63–80%); f) mCPBA, Na2HPO4, CH2Cl2, ꢀ788C (84%); g) PhMe,
2008C, sealed tube, (78%); h) DIBAL-H, CH2Cl2, ꢀ788C (87%);
i) pMBNH2, H2/Pd/C, EtOAc then (p-O2NPhO)2CO, MeCN (81%);
j) TEMPO, PhI(OAc)2, 1 mol% MsOH, CDCl3 (75%); k) MeNO2,
nBuLi, THF, RT (84%); l) Ac2O, DMAP, CH2Cl2 then NaBH4, EtOH
(67%); m) TFA (neat), reflux (80%); n) Et3OBF4, Cs2CO3, CH2Cl2
(78%). DIC=diisopropylcarbodiimide, DMAP=4-dimethylaminopyri-
dine, HMDS=hexamethyldisilazide, Ac=acetyl, mCPBA=3-chloroper-
oxybenzoic acid, DIBAL-H=diisobutylaluminum hydride, pMB=para-
methoxybenzyl, TEMPO=tetramethylpiperidine-1-oxyl, MsOH=meth-
anesulfonic acid, TFA=trifluoroacetic acid. Boc=tert-butoxycarbonyl.
the hydroxymethyl group, thus affording good yields of the
sensitive ureido aldehyde 9.[11]
Intrigued by the possibility of conducting a reductive
guanidinylation sequence and simultaneously unmasking the
uracil unit, we first synthesized the dibenzyloxypyrimidine
aldehyde 12 (Scheme 3): A slightly modified procedure
permitted the substitution of 2,4,6-tribromopyrimidine (11)
with benzyl ether groups,[12] and formylation of the lithiated
pyrimidine with dimethylformamide (DMF) afforded 12 in
good yield. Treatment of 10 with 12 in the presence of acetic
anhydride and excess cesium fluoride couples these two units
directly and allows dehydration to occur in a single operation,
thus affording 13 in 67% yield. This nitroalkene was produced
as a single geometric isomer, which is presumed to be the
E isomer from studies of the NOE interactions of the
pyrimidine proton. Attempts to reduce the nitroalkene
directly to the corresponding saturated amine led predom-
inantly to hydrolysis of the presumed enamine intermediate.
To obviate this reactivity, sodium borohydride was
employed to reduce 13 to the nitroalkane. Hydrogenolysis
of this mixture reduced the nitroalkene, thus effecting
reductive guanidinylation, and cleanly cleaved the benzyl
ether groups to afford the uracil/guanidine compounds in an
approximate 1:1 mixture of isomers. Brief exposure of this
mixture to HCl facilitated the removal of the acetate group at
C12 to give 14 and 15. The configuration of 15 was ultimately
determined by X-ray crystallographic analysis.[13] It should be
noted that the guanidine moieties were obtained as trifluor-
oacetate salts after purification by HPLC. This method of
purification has been used in all of the previous syntheses of
2.[6b,14] The small amount of synthetic material produced did
not allow this counterion to be detected by 13C NMR
spectroscopy, but it is presumed to accompany the non-
zwitterionic guanidines purified by HPLC with trifluoroacetic
acid (TFA) in the eluent.
Scheme 3. a) BnOH, nBuLi, THF/DMF (80%); b) nBuLi, Et2O, DMF,
then 10% HCl (73%); c) 12, CsF, Ac2O, MeCN (67%); d) NaBH4,
EtOH then Pd(OH)2, H2, 5% AcOH/MeOH; e) HCl (conc.), reflux for
0.5 h (14=33%, 15=30%; 2 steps); f) SO3·py, DMF, 3-ꢀ MS
(16=33% from 13, 17=33% from 13). Bn=benzyl, py=pyridine,
MS=molecular sieves.
3880
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Chemie
Ohtani, M. F. Watanabe, K. I. Harada, E. Ito, M. Watanabe,
Table 1: Inhibition of protein synthesis in rat hepatocytes.
Toxicon 1994, 32, 833.
[10] U. Kazmaier, Angew. Chem. 1994, 106, 1046; Angew. Chem. Int.
Ed. Engl. 1994, 33, 998.
[11] A. De Mico, R. Margarita, L. Parlanti, A. Vescovi, G. Piancatelli,
J. Org. Chem. 1997, 62, 6974.
[12] R. F. Schinazi, W. H. Prusoff, J. Org. Chem. 1985, 50, 841.
[13] CCDC-258351 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.ca-
m.ac.uk/data_request/cif.
Compound
Conc. [mm][a]
Protein [%][b]
GSH [%][c]
1
0.20
1.03
1.55
2
5
10
58
15
7
64
28
8
100
80
60
100
95
45
16
17
40
80
73
56
100
100
[14] a) G. R. Heintzelman, W. K. Fang, S. P. Keen, G. A. Wallace,
S. M. Weinreb, J. Am. Chem. Soc. 2001, 123, 8851; b) G. R.
Heintzelman, W. K. Fang, S. P. Keen, G. A. Wallace, S. M.
Weinreb, J. Am. Chem. Soc. 2002, 124, 3939; c) J. D. White,
J. D. Hansen, J. Am. Chem. Soc. 2002, 124, 4950.
[a] Calculated from e=4600 molꢀ1 dm3 cmꢀ1 at 263 nm. [b] Measured by
the incorporation of [35S]methionine into protein (given as % of control
values). [c] Measured as nmol of GSH per mg of protein (given as % of
control values).
[15] Co-HPLC-injection of 16 and natural 7-deoxycylindrospermop-
sin (Agilent zorbax C18 column (4.6 ꢀ 150 mm) with MeOH/
TFA/H2O (0.04:0.001:1) as the elutant at 2.0 mLminꢀ1 with
monitoring at 262 nm) gave a single peak at 5.48 min.
[16] For an excellent review of structural reassignments by total
synthesis, see: K. C. Nicolaou, S. A. Snyder, Angew. Chem. 2005,
117, 1036; Angew. Chem. Int. Ed. 2005, 44, 1012.
Contrary to previously reported findings,[5] we found that
a sample of natural 7-deoxycylindrospermopsin also inhibits
protein synthesis with potency similar to the synthetic 7-
deoxycylindrospermopsin (16). These two materials were
shown by HPLC analysis to be identical, thus corroborating
the natural occurrence of 16.[15,16]
The synthesis detailed herein should cast doubt on the
notion of uracil tautomers in the purported structure of 7-
deoxycylindrospermopsin. It appears unlikely that an oxy-
genation event at C7 or C8 occurs in the metabolism of 1, 2, or
16 (to generate an enol ketone), as the C8 diastereomer 17 is
two orders of magnitude less toxic. Compounds 14 and 16
have been labeled with 13CH3NO2 to investigate their
intermediacy in the biosynthesis of 1 and 2. These studies
will be reported in due course.
Received: February 11, 2005
Published online: May 18, 2005
Keywords: alkaloids · cyanobacteria · cycloaddition · guanidine ·
.
toxicology
[1] For an excellent review of the cylindrospermopsins, see: D. J.
Griffiths, M. L. Saker, Environ. Toxicol. 2003, 18, 78; for NIH
and EPA involvement, see: http://www.epa.gov/safewater/stan-
dard/ucmr/ and http://ntp-server.niehs.nih.gov/htdocs/Results_-
status/ResstatC/M000072.html.
[2] M. T. Runnegar, C. Y. Xie, B. B. Snider, G. A. Wallace, S. M.
Weinreb, J. Kuhlenkamp, Toxicol. Sci. 2002, 67, 81.
[3] M. T. Runnegar, S. M. Kong, Y. Z. Zhong, J. L. Ge, S. C. Lu,
Biochem. Biophys. Res. Commun. 1994, 201, 235; M. Runnegar,
S. Lu, FASEB J. 1994, 8, A419.
[4] R. Banker, S. Carmeli, M. Werman, B. Teltsch, R. Porat, A.
Sukenik, J. Toxicol. Env. Heal. A 2001, 62, 281.
[5] R. L. Norris, G. K. Eaglesham, G. Pierens, G. R. Shaw, M. J.
Smith, R. K. Chiswell, A. A. Seawright, M. R. Moore, Environ.
Toxicol. 1999, 14, 163.
[6] a) R. E. Looper, R. M. Williams, Tetrahedron Lett. 2001, 42, 769;
b) R. E. Looper, R. M. Williams, Angew. Chem. 2004, 116, 2990;
Angew. Chem. Int. Ed. 2004, 43, 2930.
[7] R. H. Li, W. W. Carmichael, S. Brittain, G. K. Eaglesham, G. R.
Shaw, Y. D. Liu, M. M. Watanabe, J. Phycol. 2001, 37, 1121.
[8] D. L. Burgoyne, T. K. Hemscheidt, R. E. Moore, M. T. Runne-
gar, J. Org. Chem. 2000, 65, 152.
[9] a) M. T. Runnegar, S. M. Kong, Y. Z. Zhong, S. C. Lu, Biochem.
Pharmacol. 1995, 49, 219; b) K. Terao, S. Ohmori, K. Igarashi, I.
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