Phosphonoxins III: Synthesis of α-aminophosphonate analogs of antifungal polyoxins with anti-Giardia activity

Article abstract of DOI:10.1021/ol101913t

A synthesis of α-aminophosphonate analogs of polyoxins, termed phosphonoxin C1, C2, and C3, has been achieved. The key step was the addition of lithium dimethyl phosphite to the aldehyde of a protected threose derivative. α-Hydroxyphosphonate analogs C4 and C5 were also obtained by taking advantage of an unprecedented conversion of an azide to hydroxyl during treatment with hydrogen on palladium on carbon. The resulting phosphonoxin C5 inhibited the growth of an intestinal protozoan, Giardia lamblia, at low micromolar concentration.

Full text of DOI:10.1021/ol101913t

ORGANIC
LETTERS
2010
Vol. 12, No. 20
4596-4599
Phosphonoxins III: Synthesis of
r-Aminophosphonate Analogs of Antifungal
Polyoxins with Anti-Giardia Activity
Michael Staake,† Jay Chauhan,† Ding Zhou,† Aaron Shanker,‡
Atasi De Chatterjee,‡ Siddhartha Das,‡ and Steven E. Patterson*,†
Center for Drug Design, Academic Health Center, UniVersity of Minnesota,
Minneapolis, Minnesota 55455, and Department of Biological Sciences,
UniVersity of Texas at El Paso, El Paso, Texas 79968-0519
patte219@umn.edu
Received August 13, 2010
ABSTRACT
A synthesis of r-aminophosphonate analogs of polyoxins, termed phosphonoxin C1, C2, and C3, has been achieved. The key step was the
addition of lithium dimethyl phosphite to the aldehyde of a protected threose derivative. r-Hydroxyphosphonate analogs C4 and C5 were also
obtained by taking advantage of an unprecedented conversion of an azide to hydroxyl during treatment with hydrogen on palladium on
carbon. The resulting phosphonoxin C5 inhibited the growth of an intestinal protozoan, Giardia lamblia, at low micromolar concentration.
The natural products polyoxins isolated from the Strepto-
myces species are a closely related class of peptidylnucleoside
antibiotics.1 They have been shown to be potent inhibitors
of many types of fungi and parasites due to their ability to
disrupt cell envelope biosynthesis.2 For example, one such
parasite is Giardia lamblia,3 a protozoan parasite that
colonizes and replicates in the intestinal tract. It is one of
the most common causes of diarrhea in the developed world
and is not only restricted to humans but affects livestock
and companion animals, where there are an estimated 100
million cases per year worldwide.4 Therefore, the need for
effective treatment continues to this date. Polyoxins5 most
likely act by mimicking the stucture of, and thereby
competing with, the natural substrate for cell envelope
biosynthesis, uridine diphosphoryl-N-acetylglucosamine (UDP-
GlcNAc, Figure 1).6 They have exhibited activity against
† University of Minnesota.
‡ University of Texas at El Paso.
Figure 1. UDP-GlcNAc, polyoxins J and L, and phosphonoxins.
(1) (a) Brock, T. D. Biology of Microrganisms; Madigan, M. T.,
Martinko, J. M., Dunlap, P. V., Clark, D. P., Eds.; Pearson Education Ltd:
Upper Saddle River, NJ, 2003. (b) For a review on polyoxins, see: Akita,
H. Heterocycles 2009, 77, 67, and references therein.
many types of fungi7 and inhibit cell wall formation in
Entamoeba.8 However, utility of these natural products as
drugs is compromised by their poor bioavailability and
metabolic instability resulting in low efficacy evidenced by
(2) (a) Debono, M.; Gordee, R. S. Annu. ReV. Microbiol. 1994, 48, 471.
(b) Riuz-Herrera, J.; San-Blas, G. Curr. Drug Targets- Infect. Disorders
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(3) Karr, C. D.; Jarroll, E. L. Microbiology 2004, 150, 1237.
10.1021/ol101913t  2010 American Chemical Society
Published on Web 09/21/2010

high inhibitory concentrations against fungal pathogens.9
Over the years, many groups have synthesized polyoxin
analogs with the goal of obtaining compounds that would
retain the antifungal and antiparasitic activity of polyoxins
but could be more suitable as drugs.5,10
The R-amino stereocenter would be generated by reaction of a
chiral aldehyde with the anion of dimethyl phosphite14 instead of
sulfinimine with dimethyl methylphosphonate.15 We also would
use a p-bromo benzoyl instead of a benzyl protecting group to aid
in the crystallization of intermediates for the determination of
stereochemistry by X-ray crystallography. We began with the
reaction of commercially available diol 6 with one equivalent of
4-BrBz-Cl in pyridine to give predominantly monobenzoylated
product 7 (Scheme 1). Oxidation of alchohol 7 with IBX16
We recently published the synthesis of a new class of
polyoxin analogs, termed phosphonoxins, that replaced the
peptide linkage to the nucleoside with a phosphonate
linkage.11,12 Phosphonates are chemically and enzymatically
stable and many such derivatives are able to penetrate cells.13
One of the phosphonoxins we synthesized (here termed
phosphonoxin A, Figure 1) is a potent inhibitor of Giardia
trophozoite growth and cyst formation in Vitro.11 We also
synthesized phosphonoxins B1 and B2, which more closely
resemble the structure of the natural polyoxins.12
Scheme 1. Synthesis of R-Diazophosphonates
We report here the synthesis of phosphonoxins C1 (1) and
C2 (2), which differ from the phosphonoxins B only in that
they do not possess the methylene spacer between the amino
phosphorus groups; they are R- instead of ꢀ-aminophosphonates.
During the course of this synthesis we also obtained R-hy-
droxyphosphonate analogs, phosphonoxins C4 (4) and C5 (5),
by the unexpected conversion of an azide into a OH group under
catalytic hydrogenation conditions. Our approach to the syn-
thesis of the phosphonoxins C involved a similar approach to
that taken to synthesize phosphonoxins B.12
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generated aldehyde 8. This aldehyde was then treated with the
lithium anion of dimethyl phosphite17 to produce R-hydroxyphos-
phonate diastereomers 9a and 9b in a 3:2 ratio in 65% combined
yield. These isomers were readily separated by column chroma-
tography. Both isomers, 9a and 9b, were then independently
converted to the corresponding azides,18 10a and 10b, via Mit-
sunobu substitution19 or triflation of the hydroxyl group followed
by nucleophilic displacement with azide.
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produce (R)-sulfinimine 11 in low yield due to cleavage of
the benzoate moiety (Scheme 2). This sulfinimine was then
reacted with the lithium anion of dimethylphosphite to give
phosphonate 12a with high selectivity (90% de) over its
diastereomer 12b. The absolute configuration of 12a was
confirmed by X-ray crystallography.
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Org. Lett., Vol. 12, No. 20, 2010
4597

Scheme 2
.
R-Diazo Stereocenter Confirmation of 10b
Scheme 3. Uridine Phosphonoxin Intermediates
Converting sulfinamide 12, of known stereochemistry, to
amine 13 by acid catalyzed cleavage of the sulfinyl group
followed by diazo transfer with TfN3 and catalytic CuSO422
gave an R-aminophosphonate identical in all aspects to 10b.
As the diazo transfer is known to proceed with retention of
configuration,22 the structure of 10b was determined as depicted.
The azides 10a and 10b were next reacted independently with
LiBr at 70 °C to cleave one of the phosphonate methyls23 giving
monomethyl esters 14a and 14b, respectively (Scheme 3). The
resulting phosphonic acids were then coupled with 2′,3′-O-
isopropylidineuridine under Mitsunobu conditions,24 followed
by removal of the remaining phosphonate methyl by again
heating in the presence of LiBr to give uridine phosphonates
15a and 15b. The benzoate ester was cleaved by heating at 50
°C in methanolic ammonia, resulting in 16a and 16b. Carbam-
ates 17a and 17b were produced by treating 16a and 16b with
trichloroacetyl isocyanate followed by ammoniolysis of the
resulting trichloroacetyl group.25 Phosphonoxin C2 was finally
obtained via removal of the acetonide group of compound 17b
by heating in 80% AcOH (Scheme 4) followed by the reduction
of the azide under Staudinger conditions26 to give uridine amine
2 in 34% yield.
Scheme 4. Phosphonoxin C2
To avoid the low yields of the Staudinger reduction, the
azide compounds 16a and 17a were instead reduced by
hydrogenation with catalytic 5% Pd on BaSO4 (Scheme 5)
in methanol to give amines 19 and 21 as the major products
in 72% and 48% yields, respectively. Removal of the
acetonide protecting group of each by heating in 80% acetic
acid in water gave phosphonoxin C3 (3), lacking the
carbamate moiety, and phosphonoxin C1 (1). To our surprise,
the palladium mediated azide reduction reaction also gave
significant amounts (16% and 34% yields) of methyl R-hy-
droxyphosphonates 20 and 22 from 16a and 17a, respectively
(Scheme 5). We investigated this process further and found
that the reaction requires hydrogen gas to be present, as no
reaction occurs with 16a and 17a when only the palladium
catalyst is present. The identities of 20 and 22 were confirmed
by cleaving the methyl ester with LiBr to obtain 23 and 24,
each with 94% diastereomeric purity, then synthesizing 23 by
an alternate route from 9a (Scheme 6).
Protection of the free OH group of 9a as an acetate group,
coupling with 2′,3′-isopropylidineuridine followed by ammonia
deprotection gave 23. Compound 23 obtained via this alternate
route was identical to major isomer 23 obtained from 20.
(22) Nyffeler, P. T.; Liang, C. H.; Koeller, K. M.; Wong, C.-H. J. Am.
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Org. Lett., Vol. 12, No. 20, 2010

Scheme 5. Phosphonoxins C1, C3, C4, and C5
Scheme 7. Proposed Mechanism of Azide to Alcohol Conversion
collapse to an R-ketophosphonate and stereoselective reduction
would give the observed product 20.29
Table 1. Inhibition of Giardia lamblia Trophozoite Growth
compound
IC50 (µM)
Phosphonoxin C1 (1)
Phosphonoxin C2 (2)
Phosphonoxin C3 (3)
Phosphonoxin C4 (4)
Phosphonoxin C5 (5)
>20
>20
>20
>20
2.3
The phosphonoxins were examined for their inhibition of
Giardia lamblia growth in culture (Table 1).30 To our
surprise, phosphonoxin C5 (5) obtained from the unique
conversion of azide to alcohol was by far the most active,
with an IC50 of 2.3 µM.
Scheme 6. R-Hydroxy Stereocenter Confirmation of 23
In conclusion, we have synthesized three novel R-amino-
phosphonate analogs of polyoxins, which we termed phospho-
noxins C1, C2 and C3. In addition, we synthesized two
R-hydroxyphosphonate analogs, C4 and C5, from the co-
products of the azide reduction, the latter of which inhibited
Giardia trophozite growth. The scope and mechanism of the
rearrangement that results in the coproducts is still under
investigation. Further chemical and biological studies of the
phosphonoxins and the R-hydroxyphosphonates will be
described in due course.
Removal of the acetonide groups of 23 and 24 gave R-hy-
droxyphosphonate analogs, phosphonoxin C4 (4) and C5 (5)
(Scheme 5). To our knowledge, this is the first example of
conversion of an azide to an alcohol by catalytic hydrogenation.
In the treatment of azide 16a with H2 and Pd on BaSO4 we
propose the methyl phosphonate ester product 20 is consistent
with a reactive intermediate involving the phosphonate moiety
(Scheme 7). Imine intermediates such as structure 27 have been
proposed for catalytic hydrogenation of azides27 and oxaphos-
phiranes similar to proposed intermediate 28 have been
described in other reaction pathways.28 Attack of methanol on
such an intermediate should give heminaminal 29, which after
Acknowledgment. This research was funded by the
Center for Drug Design of the Academic Health Center,
the University of Minnesota. S.D. was supported by the
National Institutes of Health grants S06GM081200812 and
2G12RR008124. We thank Dr. Victor G. Young Jr. for
obtaining the X-ray crystallography data.
Supporting Information Available: Experimental pro-
cedures, spectral data for all compounds and X-ray data for
compound 12. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL101913T
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