Stereoselective synthesis of novel uracil polyoxin C conjugates as substrate analogues of chitin synthase

Article abstract of DOI:10.1021/jo702564y

(Chemical Equation Presented) Stereoselective syntheses of both the natural (C5′-S) and unnatural (C5′-R) diastereoisomers of uracil polyoxin C methyl ester have been developed. The key stereocontrolled step involves nucleophilic addition of trimethylsily

Full text of DOI:10.1021/jo702564y

Stereoselective Synthesis of Novel Uracil Polyoxin C Conjugates as
Substrate Analogues of Chitin Synthase
Andrew Plant,*,‡ Peter Thompson,† and David M. Williams*,†
Centre for Chemical Biology, Richard Roberts Building, Department of Chemistry, UniVersity of Sheffield,
Sheffield S3 7HF, U.K., and Herbicide Chemistry, Syngenta, Jealott’s Hill International Research Centre,
Bracknell, Berkshire RG42 6EY, U.K.
d.m.williams@sheffield.ac.uk; andrew.plant@syngenta.com
ReceiVed December 14, 2007
Stereoselective syntheses of both the natural (C5′-S) and unnatural (C5′-R) diastereoisomers of uracil
polyoxin C methyl ester have been developed. The key stereocontrolled step involves nucleophilic addition
of trimethylsilyl cyanide to the appropriate chiral sulfinimine derived from 2′,3′-protected 5′-formyluridine
and (S)-(-)-tert-butanesulfinamide or (R)-(+)-tert-butanesulfinamide, respectively. A variety of substrate
mimics designed to function as inhibitors of chitin synthase have been synthesized by conjugation of the
methyl ester of uracil polyoxin C (UPOC) with activated isoxazole carboxylic acids. Amide bond formation
was accomplished via coupling of the amino functionality of UPOC methyl ester with a free isoxazole
acid using HBTU or alternatively an isoxazole pentafluorophenyl ester. The substrate mimics incorporate
features of the nucleoside-peptide antibiotics, the polyoxins and the nikkomycins, as well as features of
the transition state structure expected during polymerization of the natural chitin synthase substrate uridine
diphosphoryl-N-acetylglucosamine (UDP-GlcNAc), namely, a metal-binding site and glycosyl oxocar-
benium ion mimic.
Introduction
thases (CS) are attractive targets for the pharmaceutical and
agrochemical industries due to their essential role in chitin
biosynthesis in fungi and insects and due to their absence in
mammals and plants.1 Because CS are large integral membrane
proteins, there has been limited progress toward the elucidation
of detailed structural information that would be of benefit for
the rational design of inhibitors. Furthermore the exact mecha-
nistic details of chitin formation are still unresolved. Indeed the
Chitin is a homopolymer consisting of ꢀ-1,4-linked N-acetyl-
D-glucosamine (GlcNAc) units. It is one of the most abundant
natural polymers and is an essential structural component of
the insect exoskeleton and the cell walls of fungi. The
biosynthesis of chitin occurs by the polymerization of UDP-
N-acetylglucosamine (UDP-GlcNAc) (Figure 1). Chitin syn-
† University of Sheffield.
‡ Syngenta.
(1) Cohen, E. Ann. ReV. Entomol. 1987, 32, 71–93.
3714 J. Org. Chem. 2008, 73, 3714–3724
10.1021/jo702564y CCC: $40.75 2008 American Chemical Society
Published on Web 04/24/2008

Synthesis of NoVel Uracil Polyoxin C Conjugates
FIGURE 2. Postulated transition state for chitin synthesis.
have included its C1-methylenephosphonate12 and related
compounds in which the diphosphate moiety has been replaced
by nonhydrolyzable linkages13–15 such as tartrate and malonate.
The Aggarwal group described the (+)-carbocyclic uracil
polyoxin C,16 for which the furanosyl oxygen of uracil polyoxin
C is replaced by a methylene group. Such analogues display an
enhanced stability of the glycosidic linkage and may be more
amenable to cellular uptake. More recently, Miller and co-
workers have provided an alternative synthesis for this ana-
logue17 and have also prepared its C5′-epimer.18 Other analogues
that have produced comparable activity to the nikkomycins and
polyoxins as inhibitors of fungal CS have included an aza-
sugar,19 which might mimic the putative oxocarbenium ion
transition state that ensues during glycosyl transfer. Recent
studies from the Finney group have focused on designing
dimeric analogues that assume a two-active-site mechanism for
chitin synthase.14,15,20 Although at present these latter studies
have not provided unambiguous evidence for a two-active-site
mechanism, it was demonstrated that dimeric analogues (that
contain two uridyl moieties) displayed activity approximately
10-fold higher than that of corresponding monomeric analogues.
Design of Novel CS Inhibitors as Potential Pesticidal
Agents. Our design of analogues that could function as inhibitors
of CS in vivo focused on structural features of the nikkomycins,
polyoxins, and the natural substrate UDP-Glc-NAc and also
aimed to address the issues limiting in vivo activity. Specifically,
we aimed to address problems of poor cellular uptake and
bioavailability of such analogues while retaining the biologically
important feature of the natural uracil polyoxin C. On the
presumption of a transition state of the type shown in Figure 2,
we envisaged three key features required of such compounds,
namely, a uridyl component (preferably the polyoxin C nucleus),
a metal-binding site, and a suitable surrogate of the oxocarbe-
nium ion of the glycosyl unit.21 Our strategy is unique in
attempting to facilitate cellular uptake by synthesizing lipophilic
FIGURE 1. Synthesis of chitin by CS.
CHART 1. Selected Nikkomycins and Polyoxins
opposed orientations of the adjacent sugar units have prompted
suggestions that CS might possess two active sites.2
Two major families of naturally occurring nucleoside-peptide
antibiotics, the nikkomycins and the polyoxins (examples shown
in Chart 1) possess some of the structural features of the natural
substrate UDP-GlcNAc. Typically synthetic analogues of the
nikkomycins or the polyoxins have been evaluated using fungal
CS derived from Candida albicans and/or Saccharomyces
cereVisiae and have highlighted several important structural
requirements associated with biological activity. Features of
particular note include the pyrimidine base,3,4 the C5 carboxyl
group and associated C5-(S)-configuration,5–8 and the presence
of a free NH on the amide backbone of the side chain.9,10
Attempts to enhance the cellular uptake of analogues by
incorporation of hydrophobic side chains have had some
success,11 while nonhydrolyzable analogues of UDP-Glc-NAc
(2) Saxena, I. M.; Brown, R. M.; Fevre, M.; Geremia, R. A.; Henrissat, B.
J. Bacteriol. 1995, 177, 1419–1424.
(12) Chang, R.; Vo, T. T.; Finney, N. S. Carbohydr. Res. 2006, 341, 1998–
2004.
(3) Azuma, T.; Isono, K. J. Chem. Soc., Chem. Commun. 1977, 159–160.
(4) Azuma, T.; Isono, K. Chem. Pharm. Bull. 1977, 25, 3347–3353.
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1748.
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Lett. 2003, 13, 1713–1716.
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(15) Yeager, A. R.; Finney, N. S. Bioorg. Med. Chem. 2004, 12, 6451–6460.
(16) Aggarwal, V. K.; Monteiro, N. J. Chem. Soc., Perkin Trans. 1 1997,
17, 2531–2537.
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J. Org. Chem. Vol. 73, No. 10, 2008 3715

Plant et al.
(5′-(S)- configuration) were observed. In contrast, one of the
more stereoselective routes to UPOC has been described by
Barrett.30 Thus, nucleophilic addition of potassium trimethyl-
silonate to the least hindered face of the Z-nitro alkene (Scheme
1, route B) gave the (S)-R-hydroxy thioester with a dr of greater
than 15:1. However, a further six steps were required to produce
uracil polyoxin C.
As the key step in an alternative synthesis (route C, Scheme
1), Evina and Guillerm31 performed the asymmetric dihydroxy-
lation of 5′-deoxy-5′-methylene uridine (derived from the
corresponding 5′-aldehyde) using AD-mix-R with high diaste-
reoselectivity (de 95%). The product was then converted to an
O2,5′-cyclonucleoside which was subsequently treated with
azide in HMPA. A further four steps afforded UPOC in 6%
overall yield. Another approach32 to the synthesis of UPOC has
made use of R-L-talofuranosyluronic acid as the key intermedi-
ate. This in turn was obtained following the stereocontrolled
nucleophilic addition of vinylmagnesium bromide to the re face
of the protected ribose 5-aldehyde (obtained in 3 steps from
D-ribose) as shown in Scheme 1 (route D). However, diaste-
reoselectivity was limited, with optimized conditions giving a
dr of only 3.7:1. Furthermore, the carbohydrate product requires
five further synthetic steps for conversion into UPOC. More
recently,35 the Finney group has employed the same ribosyl
aldehyde which undergoes the high yielding and stereospecific
conversion to the acetylenic alcohol (route E, Scheme 1) upon
reaction with a zinc acetylide in the presence of the chiral ligand
(-)-N-methyl ephedrine. A further four steps were required to
produce the azide (common to route D) as a precursor to UPOC.
FIGURE 3. Target analogues for CS inhibition.
pro-pesticides22–24 in which the polar metal binding site is
masked within an isoxazole ring. Cleavage of the heterocycle
reveals the 1,3-dicarbonyl functionality, which is capable of
metal binding (Figure 3). This strategy has been shown to
improve transmembrane permeability and root uptake of the
herbicide isoxaflutole in planta.25 Edmunds et al. have also
reported that certain 3-alkylsulfinyl-4-nicotinoyl isoxazoles
display herbicidal activity, presumably resulting from in vivo
conversion to the corresponding diketonitriles.26 Furthermore
we sought to exploit the hypothesis that certain nicotinic acid
derivatives are taken up into plants by active transport mech-
anisms.27
Efficient Stereoselective Route to Polyoxin C. Although
several syntheses of uracil or thymine polyoxin C have been
reported previously,28–35 these either require a large number of
synthetic steps or show poor stereoselectivity (Scheme 1). One
of the shortest of these routes employed a modified Strecker
reaction between 2′,3′-isopropylidene-protected uridine 5′-
aldehyde, trimethylsilyl cyanide (TMSCN), and an amino acid
in the presence of the Lewis acid BF3 (route A, Scheme 1).29
The 5′-R-aminonitrile nucleosides obtained were transformed
to polyoxin C analogues following hydrolysis of the nitrile
function. Depending on the amino acid used, diastereomeric
ratios between 6:5 and 4:1 in favor of the natural polyoxin C
Davis et al. have shown that nucleophilic addition of Grignard
reagents to p-toluenesulfinylimino esters in the presence of a
Lewis acid is highly stereoselective (dr 83:17 to 99:1) and
following acid hydrolysis provides excellent yields of R-amino
acids.36 The mechanistic rationale for the control of the
stereochemistry in these reactions is based on the selective
coordination of the Lewis acid to the sulfinamide oxygen which
shields one face of the imine toward nucleophilic attack. This
methodology has also been applied to the stereoselective
synthesis of polyoxamic acids in which HCN addition to the
p-toluenesulfinylimines using ethylaluminium cyanoisopro-
poxide affords R-amino nitriles with a de of 91%.37 More
recently, examples of this chemistry have also been exploited
by others,38 and the addition of trimethylsilyl cyanide (TMSCN)
to sulfinimines in the presence of Lewis bases has also been
achieved with moderate-to-good stereoselectivity.39,40 A solvent-
controlled asymmetric Strecker reaction has recently been
described for the synthesis R-trifluoromethylated amino acids
using TMSCN addition to N-tert-butylsulfinylimines41 In this
case, the addition of TMSCN to sulfinylimines was investigated
in various solvents, with hexane giving the most successful
results (dr up to 99:1). In the nucleoside area, Jung et al.42 have
exploited the stereoselective addition of MeMgBr to a N-tert-
(22) Fukuto, T. R.; Fahmy, M. A. H. In Sulfur in Pesticide Action and
Metabolism; Rosen, J. D.; Magee, P. S., Cassida, J. E., Eds.; American Chemical
Society: Washington DC, 1981; ACS Symposium Series 158, pp 35-49.
(23) Fukuto, T. R. In Propesticides; Mager, P. S., Kohn, G. K., Eds.;
American Chemical Society: Washington DC, 1984; ACS Symposium Series
255, pp 87-101.
(24) Casida, J. E. In IUPAC Pesticide Chemistry:Human Welfare and
EnVironment; Miyamotot, J., Kearney, P. C., Eds.; Pergamon: New York, 1983;
pp 239-246.
(25) Pallett, K. E.; Cramp, S. M.; Little, J. P.; Veerasekaran, P.; Crudace,
A. J.; Slater, A. E. Pest Manage. Sci. 2001, 57, 133–142.
(26) Edmunds, A.; Seckinger, K.; Luethy, C.; Kunz, W.; De Mesmaeker,
A.; Schaetzer, J. Pyridine ketones useful as herbicides. WO Patent 2000/015615,
March 23, 2000.
(27) Hawkes, T. R.; Thomas, S. E. In Biosynthesis of Branched Chain Amino
Acids; Barak, Z., Chipman, D. M., Schloss, J. V., Eds.; VCH: Weinheim, 1990;
pp 373-389.
(28) Moffatt, J. G.; Damodaran, N. P.; Jones, G. H. J. Am. Chem. Soc. 1971,
93, 3812–3813.
(36) Davis, F. A.; McCoull, W. J. Org. Chem. 1999, 64, 3396–3397.
(37) Davis, F. A.; Mohanty, P. K. J. Org. Chem. 2002, 67, 1290–1296.
(38) Hansen, D. B.; Starr, M. L.; Tolstoy, N.; Joullie, M. A. Tetrahedron:
Asymmetry 2005, 16, 3623–3627.
(29) Fiandor, J.; Garcialopez, M. T.; Delasheras, F. G.; Mendezcastrillon,
P. P. Synthesis 1987, 978–981.
(30) Barrett, A. G. M.; Lebold, S. A. J. Org. Chem. 1990, 55, 3853–3857.
(31) Evina, C. M.; Guillerm, G. Tetrahedron Lett. 1996, 37, 163–166.
(32) Akita, H.; Uchida, K.; Chen, C. Y.; Kato, K. Chem. Pharm. Bull. 1998,
46, 1034–1038.
(39) Li, B. F.; Yuan, K.; Zhang, M. J.; Wu, H.; Dai, L. X.; Wang, Q. R.;
Hou, X. L. J. Org. Chem. 2003, 68, 6264–6267.
(40) Takahashi, E.; Fujisawa, H.; Yanai, T.; Mukaiyama, T. Chem. Lett. 2005,
34, 604–605.
(33) Kato, K.; Chen, C. Y. Synthesis 1998, 10, 1527–1533.
(34) Harding, K. E.; Southard, J. M. Tetrahedron: Asymmetry 2005, 16, 1845–
1854.
(41) Wang, H.; Zhao, X. M.; Li, Y. H.; Lu, L. Org. Lett. 2006, 8, 1379–
1381.
(42) Jung, P. M. J.; Beaudegnies, R.; De Mesmaeker, A.; Wendeborn, S.
Tetrahedron Lett. 2003, 44, 293–297.
(35) More, J. D.; Finney, N. S. Synlett 2003, 1307–1310.
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Synthesis of NoVel Uracil Polyoxin C Conjugates
SCHEME 1. Selected Literature Routes to Uracil Polyoxin C (UPOC)
butylsulfinylimine derivative of 2′-deoxyuridine-5′-aldehyde
to prepare the corresponding 5′-amino-5′-(S)-methyl uridine
analogue.
Herein we report an efficient and highly stereoselective
synthesis of both diastereoisomers of the methyl ester of UPOC
in which the key stereoselective step involves the Lewis acid
catalyzed addition of cyanide ion to an N-tert-butylsulfinylimine
derivative of uridine. The further elaboration of the methyl ester
of UPOC to potential CS inhibitors and the biological evaluation
of these compounds in vivo is also reported.
nucleoside 3a with the RS,R configuration was formed with a
dr of 97:3 as determined following analysis by H NMR and
1
was isolated in 77% yield following silica chromatography. The
same route using the enantiomeric (S)-(-)-tert-butanesulfina-
mide 2b was also successfully applied to the synthesis of
nucleoside 3b with the SS,S configuration which was obtained
in 70% yield and formed with a dr of 94:6. Treatment of the
R-amino nitriles 3a or 3b with HCl in methanol produced the
corresponding UPOC methyl esters with concomitant removal
of the isopropylidene protecting groups and the Ellman’s
auxiliary. Due to their polar nature, compounds 4a and 4b were
not easily purified but instead were isolated as crude products
(following neutralization and ether wash) that were used directly
in the conjugation reactions described below. The synthesis of
both the natural (C5′-(S)) and unnatural diastereoisomers of the
UPOC methyl ester allowed the assignment of C5-stereochem-
istry based on literature data28 for UPOC (i.e., the free carboxylic
acid derivative). Hence the signals for H6, H5, and H1′ all
appear downfield in the 1H NMR of the natural diastereoisomer.
Results and Discussion
Efficient Stereoselective Route to Polyoxin C. The reaction
of the aldehyde 143 with (R)-(+)-tert-butanesulfinamide in dry
dichloromethane containing anhydrous copper sulfate afforded
the sulfinimine nucleoside 2a, isolated exclusively as its
E-isomer in 88% yield (Scheme 2). Nucleoside 2a was then
dissolved in dichloromethane and BF3-etherate added at -78
°C. After 30 min TMSCN was added dropwise, and the solution
then allowed to warm to room temperature. The desired
(43) More, J. D.; Finney, N. S. Org. Lett. 2002, 4, 3001–3003.
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Plant et al.
SCHEME 2. Stereoselective Syntheses of Methyl Esters of (R) and (S)-Polyoxin C
The observed stereocontrol during the addition of cyanide to
the sulfinimines is consistent with the model proposed by Davis
et al.36 in which the sulfinyl oxygen is coordinated to the Lewis
acid, while we assume that there is minimum induction from
the stereogenic centers of the ribose. This interaction with the
sulfinimine (for the Rs isomer) directs the addition of cyanide
from the least hindered Re face of the imine to give the Cram
product (Figure 4). Interestingly, the sulfinimine 5, (Figure 4)
when reacted with TMSCN in the presence of boron trifluoride,
afforded the corresponding R-amino nitrile 6 (Figure 4) with
little or no stereocontrol (Figure 4). Thus, two diastereomeric
products were obtained, which were separated by silica column
chromatography, the faster- and slower-eluting products (6a and
6b, respectively) being isolated in a ratio of 2:3. Although the
bulkier protecting groups on the sugar and the presence of the
benzyl group on the pyrimidine of 5 may be significant, we are
uncertain of the exact reason for the loss of stereocontrol during
this reaction.
Synthesis of Isoxazole Analogues for Conjugation to
UPOC Methyl Ester. The structures in which we were
interested required the syntheses of isoxazole-4-carboxylic acid
analogues bearing a 3-H, Br, or MeS substituent and a tethered
basic group at C5. These were synthesized using either 1,3-
dipolar cycloaddition chemistry or reaction of a three-carbon
unit with hydroxylamine (Scheme 3). Thus treatment of the
alkyne 7 with oxime 8 as described by Moore et al.44 afforded
the desired isoxazole 9 in 77% yield. Deprotection of 9 gave
the corresponding alcohol 10 which was then mesylated to afford
11. Displacement of the mesylate of 11 by 2-mercaptopyridine
or 2-mercaptoimidazole gave 12 and 13, respectively, both in
excellent yield. These were subsequently debenzylated using
BCl3 to give the corresponding carboxylic acids 14 and 15.
Isoxazoles bearing a 3-H or 3-SMe substituent were prepared
by reaction of a three-carbon unit with hydroxylamine. Thus
compound 16, obtained in turn from methyl 4-chloroacetoacetate
and triethylorthoformate, was reacted with hydroxylamine to
give the isoxazole 17 in 75% yield as a single regioisomer. Our
attempts to introduce substituents via SN2 displacement of
chloride from 17 using nucleophiles such as azole-derived anions
or a variety of amines were all unsuccessful, instead leading to
base-mediated ring opening of the isoxazole (as shown in Figure
3). Indeed the only types of nucleophile that resulted in
displacement of chloride rather than ring opening were thiols.
Thus we successfully prepared isoxazole 18 in 74% yield using
2-mercaptopyridine as the nucleophile. Acid hydrolysis of 18
gave the carboxylic acid 19. When we repeated the reaction
with 2-mercaptoimidazole, acid hydrolysis of the product to
afford the carboxylic acid resulted in a zwitterion that proved
difficult to purify. Consequently we first converted the isoxazole
17 to the corresponding carboxylic acid 20 under acidic
conditions and then displaced the chloride with 2-mercaptoimi-
dazole. The product 21 precipitated from the solution and
required no further purification. The reaction of hydroxylamine
with the ketene dithioacetal 22, derived from methyl nicotiny-
lacetate, gave the MeS-substituted isoxazole 23 in 81% yield,
FIGURE 4. Proposed origin of stereocontrol during cyanide addition
to sulfinimines.
(44) Moore, J. E.; Goodenough, K. M.; Spinks, D.; Harrity, J. P. A. Synlett
2002, 2071–2073.
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Synthesis of NoVel Uracil Polyoxin C Conjugates
SCHEME 3. Syntheses of Isoxazole Carboxylic Acids
again as the only regioisomer. Basic hydrolysis of 23 gave the
carboxylic acid 24.
due to the short reaction time (30 min) and low pKa of the
pentafluorophenol. Attempts to react 29 with UPOC methyl ester
initially failed using DMF as solvent. A study of the stability
of 29 revealed that ring opening was essentially complete after
simply stirring in anhydrous DMF or DMSO (in the absence
of any base) for 24 h. However, the compound was stable in
dioxane, acetone, acetonitrile, and methanol. Indeed both (S)-
and (R)-diastereoisomers of UPOC methyl ester (4a and 4b,
respectively) could be successfully acylated with 29 in methanol
to give the products 30 and 31 in 30% and 39% yield (two
steps from 3a or 3b), respectively. Attempts to prepare the PfP
ester of 15 were unsuccessful leading to a number of products
such as those from ring opening and as evidenced by MS, the
formation of an acyl imidazolium ion derived from acylation
of the imidazole ring by the pentafluorophenyl ester. In contrast,
preparation of the pentafluorophenyl ester 32 was possible,
which in turn allowed the synthesis of the analogues 33 and 34
containing the 3-MeS isoxazole.
Synthesis of UPOC-Isoxazole Conjugates: Conjugation
of Isoxazole Analogues to UPOC Methyl Ester. Initially we
attempted to react the UPOC methyl ester 4a with isoxazole
19 following its activation as the N-hydroxysuccinimidyl (NHS)
ester using DCC. Although the NHS ester formation occurred,
this coincided with isoxazole ring opening as shown by MS
analysis and the absence of the imidazole 3-H ring proton in
the NMR spectrum. As an alternative conjugation method, we
investigated the use of O-(benzotriazol-1-yl)-N,N,N′,N′-tetram-
ethyluronium hexafluorophosphate (HBTU). Thus, the 3-bro-
moisoxazole derivatives 14 and 15 both reacted with UPOC
methyl ester (S and R diastereoisomers) in the presence of
HBTU, hydroxybenzotriazole, and triethylamine in DMF. Yields
of the target nucleosides 25-28 (Scheme 4) were between 21
and 45% (for two steps from 3a/3b via 4a/4b). However, when
the same reaction was attempted using the 3-H isoxazole 19,
no reaction was observed.
Ring-Opening Reactions and Biological Screening of
UPOC-Isoxazole Conjugates. An essential design character-
istic of the synthesized UPOC-isoxazole conjugates was the
assumption that isoxazole ring opening to the corresponding
diketonitriles would take place in planta, mediated either by
mildly basic conditions (R ) H), by enzyme catalysis (R )
SMe), or alternatively, via single electron transfer processes
The propensity for ring opening of 19 in the presence of base
led us to consider alternative strategies for activation of the
carboxylic acid. Thus, we successfully prepared the pentafluo-
rophenyl (PfP) ester 29 in 66% yield following reaction of 19
with pentafluorophenol and EDC in DMF. In this case only very
limited ring opening of the isoxazole had occurred, presumably
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Plant et al.
SCHEME 4. UPOC-Isoxazole Conjugates (Yields from 3a or 3b Shown)
(R ) Br). To provide reference standards the putative pro-
pesticides 25, 30, and 33 were ring-opened by applying known
methodologies to afford the corresponding diketonitriles shown
in Figure 5.
observed negligible metabolism over a 24 h period (for
compounds 30 and 33 values of 16% and 13% respectively were
determined). HPLC and MS analysis indicated that ring opening
of the isoxazole rings had not taken place. We also evaluated
the compounds 25-28, 30, 31, 33, and 34 against a range of
commercial target and model organisms including insect pests,
fungal pathogens, and weeds using in vivo high-throughput
screening.51 Fungal species were evaluated in mycelial growth
tests in artificial media or in leaf disk assays for obligate
pathogens. The test compounds were applied prior to inoculation
or incorporated into the media. Insect species were evaluated
on artificial diet or cotton leaf disk assays, treated with test
compounds, depending on species and developmental stage.
Herbicide effects were evaluated using monocotyledonous and
dicotyledonous model species grown on artificial media amended
with the test compounds. All tests were completed in 96-well
microtiter plates and incubated under controlled conditions for
4-10 days, at which time inhibitory or lethal effects were
assessed visually and scored. We did not detect any significant
levels of biological activity for any of the compounds reported
herein. Since the biological evaluation of these compounds was
performed in whole-cell or whole-organism assays, the lack of
activity may be due to a variety of possible reasons not directly
related to the abilities of these compounds to act as CS inhibitors
or otherwise. Thus, additional factors that might preclude their
biological activity potentially include poor cellular uptake per
se. Alternatively it is feasible that following cellular uptake these
compounds may not undergo ring opening to the dicarbonyl
species and/or may be deactivated, for example, following
enzymatic hydrolysis of the amide bond. Future strategies that
might be employed include tuning the conditions for which the
isoxazole moiety undergoes ring opening and increasing meta-
As expected, compound 30 containing the 3-H isoxazole
underwent ring opening under mildly basic conditions (triethy-
lamine in methanol) to produce the cyano-1,3-diketone 35 in
75% yield.45 In contrast, under basic conditions (pH 10 buffer)
the analogues 33 and 26 underwent hydrolysis of their respective
amide bonds faster than any ring-opening reactions. However
the corresponding diketonitriles derived from these conjugates,
namely, 35 and 36, respectively, could be formed upon treatment
with iron(II) chloride, a single-electron transfer process.46,47
However the strong chelation of Fe(III) by both of these
analogues made purification and hence 1H NMR unfeasible, and
these compounds were characterized by MS only.
Physicochemical studies48,49 revealed that, as expected,
compound 30 displayed a relatively short half-life (of less than
1 h) for conversion into 35 at pH 7 and 40 °C (data not shown).
Compounds 25 (R ) Br) and 33 (R ) SMe) were stable under
these conditions. Pesticides are often applied as spray solutions
to crop plants and in order to gain a feel for the metabolic
stability of our novel pro-pesticides in an agronomically relevant
biological system we examined selected compounds in a maize
cell culture assay.50 In this assay, compounds are incubated in
maize cell culture and growth medium and at predetermined
times acetonitrile is added, the sample frozen and then thawed,
and the supernatant analyzed by HPLC and MS. In the current
study we analyzed samples for unchanged 30 and 33 and their
ring-opened products 35 and 36, respectively. Surprisingly we
(45) Schenone, P.; Fossa, P.; Menozzi, G. J. Heterocycl. Chem. 1991, 28,
453–457.
(46) Kociolek, M. G.; Straub, N. G.; Marton, E. J. Lett. Org. Chem. 2005,
2, 280–282.
(47) Kociolek, M. G.; Straub, N. G.; Schuster, J. V. Synlett 2005, 259–262.
(48) Smith, S. C.; Clarke, E. D.; Ridley, S. M.; Bartlett, D.; Greenhow, D. T.;
Glithro, H.; Klong, A. Y.; Mitchell, G.; Mullier, G. W. Pest Manage. Sci. 2005,
61, 16–24.
(49) Clarke, E. D.; Greenhow, D. T.; Adams, D. Pestic. Sci. 1998, 54, 385–
393.
(50) Seebach, D.; Kimmerlin, T.; Perry, S.; Greenhow, G.; Lind, R. Chem.
BiodiVersity 2004, 1, 1391–1400.
FIGURE 5. Diketonitrile derivatives derived from ring-opening reac-
tions of UPOC-isoxazole conjugates.
(51) Ridley, S. M.; Elliott, A. C.; Yeung, M.; Youle, D. Pestic. Sci. 1998,
54, 327–337.
3720 J. Org. Chem. Vol. 73, No. 10, 2008

Synthesis of NoVel Uracil Polyoxin C Conjugates
to 0 °C. HCl gas was then slowly bubbled through the solution for
1 h. The resulting dark red solution was then left to stir at room
temperature for 48 h. The reaction was then cooled to 0 °C, water
(10 mL) was added slowly, and the mixture was then evaporated
to give a red solid. This was dissolved in water and neutralized
with 0.1 M aqueous NaOH. The solution was then extracted with
ether and the aqueous layer evaporated to give a yellow solid that
was used without further purification (5.24 g, 76% pure). HRMS
(ESI) m/z calcd for C11H16N3O7 [M + H]+ 302.0988, found
302.0996. 1H NMR (250 MHz, DMSO-d6): δ 3.63 (s, 3H),
3.67-3.70 (m, 1H), 3.95 (dd, J ) 5.5 Hz, 2.7 Hz, 1H), 4.04-4.15
(m, 2H, H2′), 5.67 (d, J ) 7.9 Hz, 1H), 5.78 (d, J ) 6.4 Hz, 1H),
7.91 (d, J ) 7.9 Hz, 1H). 13C NMR (60 MHz, D2O): δ 53.5, 54.3,
69.2, 72.4, 82.2, 92.0, 102.6, 143.3, 151.5, 166.2, 170.5.
bolic stability using strategies that have been persued by other
groups as outlined above. In the former case, for example the
incorporation of various lipophilic ester functional groups at
the 3-position of the isoxazole might be envisaged since these
may undergo better cell uptake and following enzymatic
hydrolysis to the carboxylic acid would undergo subsequent
decarboxylation and ring opening.53
Conclusions
In summary, we have developed an efficient and highly
diatereoselective synthetic route that allows the preparation of
the either the R or S diastereoisomer of UPOC methyl ester.
We have developed methods for the conjugation of UPOC
methyl ester (R or S) to a variety of isoxazole carboxylic acids
which incorporate a basic side chain. These conjugates can
undergo ring opening under basic conditions or in the presence
of divalent metal ions to reveal a metal-binding site composed
of a diketonitrile function. As such, the compounds possess
structural features of both the transition state of the natural
substrate during chitin biosynthesis and of the natural polyoxin
C inhibitors of chitin synthase. Unfortunately, these novel
compounds lack any appreciable in vivo pesticidal activity.
Studies are ongoing to evaluate the intrinsic activity against CS
and to better understand the factors influencing bioavailability.
Benzyl 4-tert-Butyl(diphenyl)hydroxysilane-but-2-ynoate (7).
tert-Butyl(diphenyl)(prop-2-ynyloxy)silane52 (19.00 g, 64.60 mmol)
was dissolved in dry THF (100 mL) and the solution was cooled
to -78 °C under N2. Next, 2.0 M nBuLi in hexane (38.60 mL,
96.90 mmol) was added dropwise over 20 min. The reaction was
then allowed to warm to room temperature, causing a color change
from colorless to brown. The reaction was then cooled again to
-78 °C and benzyl chloroformate (13.70 mL, 96.90 mmol) in dry
THF (50 mL) was added dropwise to give a yellow solution. This
was stirred at -78 °C for 5 h and then allowed to warm to room
temperature overnight. The reaction was then quenched with
saturated aqueous NH4Cl solution (100 mL) and extracted with
ether, and the combined organic layers were dried (MgSO4) and
evaporated to a yellow oil. Flash chromatography (0-10% EtOAc
in hexane) gave a colorless oil (25.07 g, 58.57 mmol, 91%). HRMS
(ESI) m/z calcd for C27H28O3NaSi [M + Na]+ 451.1705, found
Experimental Methods
1
451.1685. H NMR (250 MHz, DMSO-d6): δ 0.98 (s, 9H), 4.54
(RS)-2-Methyl-2-propanesulfinic Acid 2′,3′-O-Isopropyluri-
dine-5′-methyleneamide (2a). Nucleoside 143 (9.05 g, 32.10
mmol), (R)-(+)-tert-butanesulfinamide (4.28 g, 35.31 mmol) and
anhydrous CuSO4 (10.25 g, 64.10 mmol) were dissolved in dry
CH2Cl2 (100 mL) and stirred at room temperature for 24 h. The
suspension was filtered using filter aid and the filtrate was
evaporated to a foam. Flash chromatography (1:1 hexane/EtOAc)
gave a white solid (10.82 g, 28.10 mmol, 88%). HRMS (ESI) m/z
(s, 2H), 5.19 (s, 2H), 7.37-7.64 (m, 15H). 13C NMR (60 MHz,
CDCl3): δ 19.2, 26.7, 52.3, 67.6, 76.6, 86.1, 127.5, 127.9, 128.4,
128.4, 128.5, 128.6, 128.7, 128.8, 130.0, 132.4, 134.9, 135.6, 153.1.
Benzyl 3-Bromo-5-(tert-butyl(diphenyl)methylhydroxysi-
lane)isoxazole-4-carboxylate (9). Alkyne 7 (4.90 g, 11.45 mmol)
and hydroxycarbonimidic dibromide 844 (2.30 g, 11.45 mmol) were
dissolved in dry DME (50 mL). KHCO3 (2.30 g, 22.90 mmol) was
then added and the resulting suspension was stirred at 50 °C
overnight under N2. The mixture was then cooled and filtered and
the filtrate was evaporated. Flash chromatography (49:1 hexane/
EtOAc) gave a colorless oil (5.62 g, 10.24 mmol, 89%). HRMS
(ESI) m/z calcd for C28H29NO4SiBr [M + H]+ 550.1049, found
1
calcd for C16H24N3O6S [M + H]+ 386.1386, found 386.1382. H
NMR (250 MHz, DMSO-d6): δ 1.13 (s, 9H), 1.31 (s, 3H), 1.49 (s,
3H), 4.88 (dd, J ) 3.2 Hz, 1H), 5.03 (dd, J ) 6.1, 3.2 Hz), 5.21
(d, J ) 6.4 Hz, 1H), 5.61 (dd, J ) 7.9, 1.8 Hz, 1H), 5.85 (s, 1H),
7.81 (d, J ) 7.9 Hz, 1H), 7.87 (d, J ) 3.4 Hz, 1H), 11.44 (s, 1H).
13C NMR (60 MHz, DMSO-d6): δ 22.1, 25.0, 26.7, 56.6, 84.1, 84.1,
89.7, 95.6, 101.5, 112.9, 144.4, 150.4, 163.4, 168.3.
1
550.1022. H NMR (250 MHz, DMSO-d6): δ 0.97 (s, 9H), 5.11
(s, 2H), 5.16 (s, 2H), 7.27-7.64 (m, 15H). 13C NMR (60 MHz,
CDCl3): δ 19.3, 26.7, 58.3, 67.0, 109.3, 127.8, 127.9, 128.3, 128.6,
128.7, 128.8, 129.9, 130.1, 132.3, 134.8, 135.5, 135.6, 135.7, 140.5,
159.4, 176.7.
(RS,R)-2-Methyl-2-propanesulfinic Acid 2′,3′-O-Isopropyl-
uridine-5′-cyanomethylamide (3a). Nucleoside 2a (3.34 g, 8.68
mmol) was dissolved in dry CH2Cl2 (100 mL) under argon. BF3OEt2
(1.65 mL, 13.02 mmol) was added and the solution was cooled to
-78 °C and stirred for 10 min. TMSCN (2.30 mL, 17.35 mmol)
was then added and the reaction was stirred at -78 °C for 2 h.
The reaction was then allowed to warm to room temperature and
was stirred for 3 h. Brine (100 mL) was added and the reaction
was stirred for 5 min. The organic layer was then washed with
brine, dried (MgSO4), and evaporated. Flash chromatography (50%
EtOAc, 50% hexane) gave a white foam (2.76 g, 6.70 mmol, 77%).
HRMS (ESI) m/z calcd for C17H25N4O6S [M + H]+ 413.1495, found
Benzyl 3-Bromo-5-(hydroxymethyl)isoxazole-4-carboxylate
(10). Isoxazole 9 (5.00 g, 9.11 mmol) was dissolved in dry THF
under N2. Triethylamine trihydrofluoride (1.47 g, 45.53 mmol) was
then added and the solution was stirred for 24 h at room temperature
and then evaporated. The residue was dissolved in EtOAc (50 mL)
and washed with saturated aqueous NaHCO3 solution, water and
brine. The organic layer was then dried (MgSO4) and evaporated
to a yellow oil. Flash chromatography (hexane to 1:1 hexane/
EtOAc) gave a colorless oil (2.27 g, 7.28 mmol, 80%). HRMS (ESI)
m/z calcd for C12H11NO4Br [M + H]+ 311.9871, found 311.9874.
1H NMR (250 MHz, DMSO-d6): δ 4.85 (s, 2H), 5.33 (s, 2H),
7.30-7.49 (m, 5H). 13C NMR (100 MHz, DMSO-d6): δ 55.5, 66.6,
108.6, 128.1, 128.4, 128.5, 128.6, 128.6, 135.8, 140.7, 159.2, 178.7.
Benzyl 3-Bromo-5-[(methylsulfonyl)oxymethyl]isoxazole-4-
carboxylate (11). Isoxazole 10 (2.00 g, 8.01 mmol) was dissolved
in dry CH2Cl2 (10 mL) under Ar and dry triethylamine (1.62 mL,
16.02 mmol) was added followed by methanesulfonyl chloride (1.83
g, 16.02 mmol). The reaction was stirred at room temperature for
1
413.1497. H NMR (250 MHz, DMSO-d6): δ 1.15 (s, 9H), 1.28
(s, 3H), 1.47 (s, 3H), 4.24 (dd, J ) 9.5, 2.4 Hz, 1H), 4.77 (t, J )
9.2 Hz, 1H), 4.92 (dd, J ) 6.1, 2.7 Hz, 1H), 5.20 (d, J ) 6.4 Hz,
1H), 5.68 (d, J ) 7.9 Hz, 1H), 5.80 (s, 1H), 6.65 (d, J ) 8.9 Hz,
1H), 7.84 (d, J ) 7.9 Hz, 1H), 11.56 (s, 1H). 13C NMR (60 MHz,
DMSO-d6): δ 20.2, 22.7, 24.6, 47.9, 55.6, 81.1, 82.7, 86.1, 96.2,
100.4, 112.7, 116.1, 143.5, 149.5, 163.4.
(S)-Uracil Polyoxin C Methyl Ester (4a). Nucleoside 3a (5.10
g, 12.38 mmol) was dissolved in dry MeOH (100 mL) and cooled
(52) Bekele, T.; Brunette, S. R.; Lipton, M. A. J. Org. Chem. 2003, 68, 8471–
8479.
(53) Perez, C.; Janin, Y. L.; Grierson, G. S. Tetrahedron 1996, 52, 987–
992.
J. Org. Chem. Vol. 73, No. 10, 2008 3721

Plant et al.
24 h, diluted with EtOAc, and then washed with saturated aqueous
NaHCO3 solution, water and brine. The organic layer was dried
(MgSO4) and then evaporated and the residue was purified by flash
chromatography (hexane to 1:1 hexane/EtOAc) to give a white solid
(2.21 g, 5.67 mmol, 71%). HRMS (ESI) m/z calcd for
C13H12NO6NaSBr [M + Na]+ 411.9466, found 411.9470. 1H NMR
(250 MHz, DMSO-d6): δ 2.40 (s, 3H), 5.13 (s, 2H), 5.37 (s, 2H),
7.35-7.51 (m, 5H). 13C NMR (60 MHz, CDCl3): δ 33.3, 67.6,
68.4, 107.6, 128.5, 128.8, 129.1, 134.6, 140.9, 159.0, 173.1.
Benzyl 3-Bromo-5-[(pyridine-2-ylthio)methyl]isoxazole-4-
carboxylate (12). Isoxazole 11 (1.00 g, 2.56 mmol) and 2-mer-
captopyridine (342 mg, 3.08 mmol) were dissolved in dry CH2Cl2
(10 mL) under argon. Triethylamine (0.42 mL, 3.08 mmol) was
then added and the solution was stirred for 24 h at room temperature
and then evaporated. The residue dissolved in EtOAc was washed
with saturated aqueous NaHCO3 solution, water and brine. The
organic layer was then dried (MgSO4) and evaporated and the
residue was purified by flash chromatography (hexane to 3:1 hexane/
EtOAc) to give a white solid (886 mg, 2.18 mmol, 85%). HRMS
(ESI) m/z calcd for C17H14N2O3SBr [M + H]+ 404.9909, found
removed under vacuum to leave an orange solid (54.50 g, 265.00
mmol, quant) as a mixture of E and Z isomers. This was used
without further purification. HRMS (ESI) m/z calcd for
C8H11O4NaCl [M + Na]+ 229.0244, found 229.0250. 1H NMR (250
MHz, DMSO-d6): δ 1.04-1.30 (m, 3H), 3.63-3.71 (m, 3H),
4.08-4.37 (m, 2H), 4.62-4.68 (m, 2H), 7.99-8.01 (m, 1H). 13C
NMR (60 MHz, DMSO-d6): δ 14.1, 15.1, 47.0, 50.0, 51.6, 51.7,
72.5, 72.9, 109.0, 112.0, 164.5, 165.0, 167.1, 167.4, 188.1, 188.2.
Methyl-5-(chloromethyl)isoxazole-4-carboxylate (17). To com-
pound 16 (25.00 g, 121.00 mmol) in MeOH (100 mL) at 0 °C was
added a solution of hydroxylamine hydrochloride (12.60 g, 182.00
mmol) and sodium acetate trihydrate (24.90 g, 183.00 mmol) in
water (100 mL). After stirring for 4 h, the mixture was extracted
with CH2Cl2 and the organic layer was dried (MgSO4) and
evaporated. Purification by flash chromatography (67% hexane, 33%
EtOAc) gave a colorless oil (19.30 g, 110.00 mmol, 60%). HRMS
(EI) m/z calcd for C6H6NO3Cl [M + H]+ 175.0036, found 175.0043.
1H NMR (250 MHz, DMSO-d6): δ 3.83 (s, 3H), 5.13 (s, 2H), 9.03
(s, 1H). 13C NMR (60 MHz, CDCl3): δ 32.5, 52.3, 110.8, 150.2,
160.9, 170.5.
1
404.9899. H NMR (250 MHz, DMSO-d6): δ 4.89 (s, 2H), 5.37
Methyl-5-[(pyridine-2-ylthio)methyl]isoxazole-4-carboxylate (18).
2-Mercaptopyridine (3.33 g, 30.00 mmol) was dissolved in dry
DMF (100 mL) and triethylamine (4.20 mL, 30.00 mmol) was
added. This solution was stirred for 10 min and then cooled to -40
°C and compound 17 (5.00 g, 28.57 mmol) dissolved in dry DMF
(20 mL) was slowly added. Stirring then continued at -40 °C for
3 h and then the mixture was allowed to warm to room temperature
over 12 h. The mixture was then worked up as described for 12
and gave a yellow oil (5.3 g, 21.14 mmol, 74%). This was used
without further purification. HRMS (EI) m/z calcd for C11H10N2O3S
[M + H]+ 250.0412, found 250.0424. 1H NMR (250 MHz, DMSO-
d6): δ 3.82 (s, 3H), 4.91 (s, 2H), 7.18 (t, J ) 6.4 Hz, 1H), 7.38 (d,
J ) 7.9 Hz, 1H), 7.56-7.72 (m, 1H), 8.44 (d, J ) 4.9 Hz, 1H),
8.93 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 23.3, 52.1, 109.5,
120.6, 122.0, 137.1, 149.5, 150.6, 155.6, 161.1, 173.5.
(s, 2H), 7.15 (ddd, J ) 7.3 Hz, 4.9 Hz, 0.9 Hz, 1H), 7.33-7.50
(m, 6H), 7.67 (td, J ) 7.3 Hz, 1.8 Hz, 1H), 8.37 (ddd, J ) 4.9 Hz,
1.8 Hz, 0.9 Hz, 1H). 13C NMR (60 MHz, CDCl3): δ 24.7, 67.2,
110.3, 120.3, 122.2, 128.5, 128.6, 128.7, 135.0, 136.4, 140.1, 149.5,
155.7, 159.8, 176.3.
Benzyl 3-Bromo-5-[(1H-imidazol-2-ylthio)methyl]isoxazole-
4-carboxylate (13). Isoxazole 11 (950 mg, 2.43 mmol) and
2-mercaptoimidazole (282 mg, 2.82 mmol) were dissolved in dry
CH2Cl2 (10 mL) under Ar. Triethylamine (0.39 mL, 2.82 mmol)
was then added and the mixture was stirred for 24 h at room
temperature. More 2-mercaptoimidazole (122 mg, 1.22 mmol) and
triethylamine (0.17 mL, 1.22 mmol) were then added and the
reaction was stirred for a further 24 h. The reaction was then worked
up as for 12. Flash chromatography (hexane to 3:1 hexane/EtOAc)
gave a white solid (887 mg, 2.25 mmol, 92%). HRMS (ESI) m/z
calcd for C15H13N3O3SBr [M + H]+ 393.9861, found 393.9853.
1H NMR (250 MHz, DMSO-d6): δ 4.53 (s, 2H), 5.23 (s, 2H), 6.96
(bs, 1H), 7.18 (bs, 1H), 7.32-7.47 (m, 5H), 12.45 (bs, 1H). 13C
NMR (60 MHz, CDCl3): δ 30.4, 67.3, 110.0, 125.1, 128.5, 128.7,
128.7, 128.9, 134.8, 136.2, 140.7, 159.4, 175.6.
5-[(Pyridine-2-ylthio)methyl]isoxazole-4-carboxylic Acid (19).
Compound 18 (5.80 g, 23.20 mmol) was dissolved in glacial acetic
acid (50 mL), concentrated HCl was added (25 mL) and the mixture
was stirred at reflux for 6 h. The reaction was poured onto ice and
neutralized to pH 8 with Na2CO3. The solution was then extracted
with EtOAc and then acidified with concentrated HCl. The product
was then extracted into EtOAc, dried (MgSO4), and evaporated to
give a white solid (2.02 g, 8.56 mmol, 37%). HRMS (EI) m/z calcd
3-Bromo-5-[(pyridine-2-ylthio)methyl]isoxazole-4-carboxylic Acid
(14). Isoxazole 12 (730 mg, 1.80 mmol) was dissolved in dry
CH2Cl2 (6 mL) and BCl3 (3.90 mL, 1 M solution in heptane) was
added slowly over 5 min. The reaction was then stirred for 1 h at
room temperature, then EtOAc was added and the product was
extracted into saturated aqueous NaHCO3 solution. The aqueous
layer was then acidified (pH 2) and extracted with EtOAc. The
organics were then dried (MgSO4) and evaporated to give a white
solid (519 mg, 1.66 mmol, 91%). HRMS (ESI) m/z calcd for
C10H8N2O3SBr [M + H]+ 314.9439, found 314.9426. 1H NMR (250
MHz, DMSO-d6): δ 4.91 (s, 2H, CH2S), 7.17 (dd, J ) 7.3 Hz, 4.9
Hz, 1H), 7.39 (dd, J ) 7.9 Hz, 0.9 Hz, 1H), 7.69 (td, J ) 7.3 Hz,
1
for C10H8N2O3S [M + H]+ 236.0256, found 236.0256. H NMR
(250 MHz, DMSO-d6): δ 4.90 (s, 2H), 7.18 (t, J ) 6.0 Hz, 1H),
7.38 (d, J ) 8.2 Hz, 1H), 7.69 (d, J ) 7.8 Hz, 1H), 8.45 (d, J )
4.9 Hz), 8.85 (s, 1H), 13.43 (s, 1H). 13C NMR (60 MHz, DMSO-
d6): δ 23.3, 110.5, 120.6, 121.9, 137.0, 149.5, 150.9, 155.8, 162.2,
173.0.
5-(Chloromethyl)Isoxazole-4-carboxylic Acid (20). Compound
17 (13.00 g, 74.3 mmol) was reacted as described for the preparation
of 19 using glacial acetic acid (30 mL) and concentrated HCl (15
mL) to afford a white solid (6.96 g, 43.22 mmol, 58%). HRMS
(ESI) m/z calcd for C5H4NO3Cl [M + H]+ 160.9879, found
1.8 Hz, 1H), 8.43 (dt, J ) 4.9 Hz, 0.9 Hz, 1H), 13.84 (bs, 1H). 13
C
NMR (100 MHz, DMSO-d6): δ 24.2, 110.5, 120.7, 122.0, 137.1,
1
160.9883. H NMR (250 MHz, DMSO-d6): δ 5.12 (s, 2H), 8.97
141.2, 149.5, 155.5, 160.9, 175.9.
(s, 1H), 13.60 (br s, 1H). 13C NMR (60 MHz, CDCl3): δ 32.4,
3-Bromo-5-[(1H-imidazol-2-ylthio)methyl]isoxazole-4-car-
boxylic Acid (15). Treatment of isoxazole 13 (750 mg, 1.90 mmol)
in CH2Cl2 (6 mL) with BCl3 (4.00 mL, 1 M solution in heptane)
as described for 14, followed by flash chromatography (EtOAc to
1:1 EtOAc/methanol) gave a white solid (579 mg, 1.90 mmol,
quant). HRMS (ESI) m/z calcd for C8H7N3O3SBr [M + H]+
303.9389, found 303.9389. 1H NMR (250 MHz, DMSO-d6): δ 4.66
(s, 2H), 7.02 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 24.7,
114.4, 124.0, 137.2, 141.4, 164.9, 171.9.
Methyl-2-(chloroacetyl)-3-ethoxyacrylate (16). Methyl-4-chlo-
roacetoacetate (30.60 mL, 265.70 mmol), triethylorthoformate
(88.40 mL, 531.40 mmol) and acetic anhydride (25.00 mL, 265.70
mmol) were refluxed together for 24 h. Volatile components were
110.3, 150.4, 166.1, 171.9.
5-[(Imidazol-2-ylthio)methyl]isoxazole-4-carboxylic Acid (21).
Isoxazole 20 (3.00 g, 18.63 mmol) was dissolved in dry DMF (5
mL), then triethylamine (2.60 mL) was added and the solution was
stirred for 5 min. The mixture was then added to a separate flask
containing 2-mercaptoimidazole (1.87 g, 18.63 mmol) dissolved
in dry DMF (5 mL). The reaction was then stirred for 24 h at room
temperature under Ar. The white precipitate that formed was
removed by filtration, washed with ice-cold MeOH and dried to
leave a white powder. No further purification was necessary. HRMS
(ESI) m/z calcd for C8H6N3O3S [M - H]- 224.0130, found
1
224.0121. H NMR (250 MHz, DMSO-d6): δ 4.57 (s, 2H), 7.08
3722 J. Org. Chem. Vol. 73, No. 10, 2008

Synthesis of NoVel Uracil Polyoxin C Conjugates
(s, 2H), 8.80 (s, 1H). 13C NMR (60 MHz, DMSO-d6): δ 27.9, 110.7,
124.4, 136.1, 150.9, 162.0, 172.3.
13C NMR (100 MHz, DMSO-d6): δ 24.1, 52.6, 54.5, 70.8, 72.0,
83.3, 88.8, 102.7, 114.9, 120.8, 122.3, 137.6, 140.5, 142.2, 149.9,
151.1, 155.9, 159.4, 163.4, 170.0, 171.4.
Methyl 3,3-Bis(methylthio)-2-(pyridin-3-ylcarbonyl)acrylate
(22). Methyl nicotinoylacetate (3.00 g, 16.76 mmol) and potassium
carbonate (2.80 g, 16.76 mmol) were dissolved in dry DMF (30
mL) and stirred for 2 h at room temperature to give a white
suspension. Carbon disulfide (3.82 g, 50.28 mmol) was then added
to give an orange precipitate. After 2 h, iodomethane (4.76 g, 33.52
mmol) was added and stirring was continued for a further 12 h.
The solution was then poured onto ice, extracted with EtOAc, dried
(MgSO4), and evaporated. Flash chromatography (1:1 hexane/
EtOAc) gave an orange solid (3.55 g, 12.53 mmol, 75%). HRMS
(ESI) m/z calcd for C12H14NO3S2 [M + H]+ 284.0415, found
Methyl-1,5-dideoxy-1-(3,4-dihydro-2,4-dioxo-1-(2H)-pyrimi-
dinyl)-5-{3-bromo-5-[(imidazol-2-ylthio)methyl]isoxazole-4-car-
bonylamino}-ꢀ-D-allofuranuronate (26). UPOC methyl ester 4a
(216 mg, 0.72 mmol), isoxazole 15 (240 mg, 0.79 mmol), HBTU
(299 mg, 0.79 mmol) and HOBt ·H2O (121 mg, 0.79 mmol) were
dissolved in dry DMF (4 mL) under Ar. Triethylamine (0.22 mL,
1.58 mmol) was then added and the reaction was stirred for 24 h
at room temperature Work-up and purification as for 25 gave a
beige-colored foam (87 mg, 0.15 mmol, 21% (from compound 3a)).
HRMS (ESI) m/z calcd for C19H20BrN6O9S [M + H]+ 587.0196,
1
1
284.0416. H NMR (250 MHz, DMSO-d6): δ 2.23 (bs, 3H), 2.53
found 587.0179. H NMR (250 MHz, DMSO-d6): δ 3.67 (s, 3H),
(bs, 3H), 3.60 (s, 3H), 7.59 (dd, J ) 7.9 Hz, 4.9 Hz, 1H), 8.20 (dt,
J ) 7.9 Hz, 2.0 Hz, 1H), 8.82(dd, J ) 4.9 Hz, 1.8 Hz, 1H), 8.98
(d, J ) 2.4 Hz, 1H). 13C NMR (60 MHz, CDCl3): δ 17.4, 19.5,
52.2, 123.7, 130.6, 132.5, 136.1, 150.6, 153.5, 159.2, 163.3, 190.2.
Methyl 3-(Methythio)-5-(pyridin-3-yl)isoxazole-4-carboxy-
late (23). To compound 22 (3.50 g, 12.37 mmol) in MeOH (20
mL) was added a solution of hydroxylamine hydrochloride (4.30
g, 61.84 mmol) and sodium acetate trihydrate (8.45 g, 61.84 mmol)
in water (20 mL). Further MeOH was then added after a precipitate
appeared. The yellow solution was stirred at room temperature for
24 h, then extracted with CH2Cl2, dried (MgSO4), and evaporated.
Flash chromatography (1:1 hexane/EtOAc) gave a yellow solid
(2.50 g, 10.02 mmol, 81%). HRMS (ESI) m/z calcd for
C11H11N2O3S [M + H]+ 251.0365, found 251.0372. 1H NMR (250
MHz, DMSO-d6): δ 2.56 (s, 3H), 3.76 (s, 3H), 7.62 (dd, J ) 7.9
Hz, 4.9 Hz, 1H), 8.28 (dt, J ) 7.9 Hz, 2.0 Hz, 1H), 8.79 (dd, J )
4.9 Hz, 1.5 Hz, 1H), 9.02 (d, J ) 2.1, 1H). 13C NMR (60 MHz,
MeOH-d4): δ 13.8, 52.5, 109.9, 125.0, 138.5, 150.3, 152.6, 162.5,
164.0, 171.7.
3-(Methylthio)-5-(pyridin-3-yl)isoxazole-4-carboxylic Acid
(24). Compound 23 (2.50 g, 10.00 mmol) was dissolved in MeOH
(37.50 mL) and a 15% (w/v) KOH solution (12.50 mL) added
slowly. The reaction was stirred for 30 min at room temperature
and then water (50 mL) was added. The basic solution was taken
to pH 10 with dilute aqueous HCl and then extracted with EtOAc
to remove unreacted starting material. The aqueous layer was then
acidifed with concentrated HCl and extracted with EtOAc, and the
combined organic layers were dried (MgSO4) and evaporated to
afford a beige-colored solid (2.10 g, 8.90 mmol, 89%). HRMS (ESI)
m/z calcd for C10H9N2O3S [M + H]+ 237.0334, found 237.0345.
1H NMR (250 MHz, DMSO-d6): δ 2.54 (s, 3H), 7.61 (dd, J ) 7.9
Hz, 4.9 Hz, 1H), 8.28 (dt, J ) 7.9 Hz, 2.0 Hz), 8.77 (dd, J ) 4.9
Hz, 1.5 Hz, 1H), 9.03 (d, J ) 2.1 Hz, 1H), 13.51 (bs, 1H). 13C
NMR (60 MHz, DMSO-d6): δ 13.1, 109.3, 122.8, 123.4, 136.8,
149.4, 151.8, 161.8, 162.4, 170.2.
4.06-4.22 (m, 3H), 4.68 (s, 2H), 4.88-4.94 (m, 1H), 5.44 (bs,
1H), 5.55 (d, J ) 5.5 Hz, 1H), 5.71 (d, J ) 7.9 Hz, 1H), 5.76 (d,
J ) 5.5 Hz, 1H), 6.96 (bs, 1H), 7.18 (bs, 1H), 7.60 (d, J ) 8.2 Hz,
1H), 10.76 (d, J ) 8.5 Hz, 1H), 11.41 (bs, 1H), 12.42 (bs, 1H).
13C NMR (100 MHz, DMSO-d6): δ 22.6, 52.6, 54.4, 71.0, 72.0,
83.0, 88.7, 102.6, 114.9, 119.5, 124.8, 138.1, 140.8, 141.4, 151.0,
158.9, 163.3, 170.2, 170.2.
Pentafluorophenyl 5-[(Pyridine-2-ylthio)methyl]isoxazole-4-
carboxylate (29). Isoxazole 19 (2.00 g, 8.47 mmol) and EDC (1.63
g, 8.47 mmol) were dissolved in dry DMF (15 mL) under Ar. A
solution of pentafluorophenol (1.72 g, 9.32 mmol) in dry DMF (5
mL) was then added slowly and the mixture was stirred at room
temperature for 30 min and then evaporated. The residue was
dissolved in acetone (100 mL), 0.01 M pH 6 phosphate buffer (50
mL) was added and the mixture was extracted into ether. The
combined organic layers were washed with water and brine, dried
(MgSO4), and evaporated. Flash chromatography (3:1 hexane/
EtOAc) afforded a white solid (2.36 g, 5.87 mmol, 69%). HRMS
(ESI) m/z calcd for C16H8N2O3F5S [M + H]+ 403.0176, found
403.0172. 1H NMR (250 MHz, DMSO-d6): δ 4.88 (s, 2H, CH2S),
6.99 (dd, J ) 7.3 Hz, 4.9 Hz, 1H), 7.17 (d, J ) 7.9 Hz, 1H), 7.48
(td, J ) 7.6 Hz, 1.8 Hz, 1H), 8.35 (d, J ) 4.9 Hz, 1H), 8.58 (s,
1H). 13C NMR (60 MHz, CDCl3): δ 24.5, 107.4, 119.0, 120.8,
122.8, 136.0, 136.9, 139.2, 140.0, 141.7, 143.2, 149.3, 150.0, 155.5,
156.7, 176.4. 19F NMR (250 MHz, acetone-d6): δ -172.1 to -171.9
(m, 1F), -166.2 to -166.0 (t, 1F), -163.4 to -163.2 (t, 1F),
-158.5 to -158.3 (t, 1F), -153.3 to -153.2 (d, 1F).
Methyl-1,5-dideoxy-1-(3,4-dihydro-2,4-dioxo-1-(2H)-pyrimidi-
nyl)-5-{5-[(pyridin-2-ylthio)methyl]isoxazole-4-carbonylamino}-
ꢀ-D-allofuranuronate (30). UPOC methyl ester 4a (1.00 g, 3.32
mmol) and isoxazole 29 (1.50 g, 3.72 mmol) were dissolved in
dry MeOH (10 mL) and stirred at room temp under Ar. After 48 h
the solvent was removed under vacuum and the residue was
dissolved in water (50 mL), which was extracted with EtOAc. The
combined organic layers were then dried (MgSO4) and evaporated.
Purification by flash chromatography (EtOAc to 4:1 EtOAc/MeOH)
gave an off-white solid (500 mg, 0.96 mmol, 30% (from compound
3a)). HRMS (ESI) m/z calcd for C21H22N5O9S [M + H]+ 520.1138,
Methyl-1,5-dideoxy-1-(3,4-dihydro-2,4-dioxo-1-(2H)-pyrimi-
dinyl)-5-{3-bromo-5-[(pyridin-2-ylthio)methyl]isoxazole-4-car-
bonylamino}-ꢀ-D-allofuranuronate (25). UPOC methyl ester 4a
(351 mg, 1.17 mmol), isoxazole 14 (400 mg, 1.28 mmol), HBTU
(486 mg, 1.28 mmol) and HOBt ·H2O (196 mg, 1.28 mmol) were
dissolved in dry DMF (5 mL) under argon. Triethylamine (0.36
mL, 2.58 mmol) was then added and the reaction was stirred for
24 h at room temperature Solvent was evaporated and the slurry
was partitioned between EtOAc and water. The aqueous layer was
further extracted with EtOAc and the combined organic layers were
dried (MgSO4) and evaporated to give a yellow foam. Flash
chromatography (EtOAc to 20:1 EtOAc/MeOH) gave an off-white
foam (315 mg, 0.54 mmol, 45% (from compound 3a)). HRMS (ESI)
m/z calcd for C21H21BrN5O9S [M + H]+ 598.0243, found 598.0231.
1H NMR (250 MHz, DMSO-d6): δ 3.65 (s, 3H), 4.08 (m, 1H),
4.20-4.27 (m, 2H), 4.74 (s, 2H), 4.95 (dd, J ) 8.2 Hz, 6.7 Hz,
1H), 5.41-5.43 (m, 1H), 5.53-5.55 (m, 1H), 5.72 (dd, J ) 7.9
Hz, 2.1 Hz, 1H), 5.78 (d, J ) 5.5 Hz, 1H), 7.13 (dd, J ) 6.4 Hz,
5.2 Hz, 1H), 7.35 (d, J ) 8.2 Hz, 1H), 7.55-7.72 (m, 2H, Py),
8.39 (d, J ) 4.9 Hz, 1H), 9.38 (d, J ) 8.2 Hz, 1H), 11.41 (bs, 1H).
1
found 520.1160. H NMR (250 MHz, DMSO-d6): δ 3.67 (s, 3H),
4.08-4.23 (m, 4H), 4.89 (s, 2H), 5.39 (d, J ) 4.9 Hz, 1H), 5.54
(d, J ) 5.2 Hz, 1H), 5.68 (d, J ) 8.2 Hz, 1H), 5.77 (d, J ) 5.8 Hz,
1H), 7.13-7.18 (m, 1H), 7.36 (d, J ) 7.9 Hz, 1H), 7.57 (d, J )
7.9 Hz, 1H), 7.68 (m, 1H), 8.43-8.46 (m, 1H), 9.05 (s, 1H),
9.10-9.13 (d, J ) 7.9 Hz, 1H), 11.43 (bs, 1H). 13C NMR (60 MHz,
MeOH-d4): δ 25.0, 53.2, 55.5, 71.6, 74.1, 84.4, 93.8, 103.2, 113.8,
121.5, 123.3, 138.2, 143.6, 150.3, 150.6, 152.3, 158.1, 163.1, 165.9,
171.1, 172.5.
Pentafluorophenyl 3-(Methylthio)-5-pyridin-3-ylisoxazole-4-
carboxylate (32). Compound 24 (1.50 g, 6.36 mmol) was dissolved
in dry DMF under argon (20 mL) and EDC (1.34 g, 7.00 mmol)
added. A solution of pentafluorophenol (1.29 g, 7.00 mmol) in dry
DMF (2 mL) was then added, the mixture was stirred overnight at
room temperature then poured onto acetone (50 mL), and 0.01 M
pH 6 phosphate buffer (50 mL) was added. The solution was then
extracted with EtOAc and the organic layers were combined, dried
J. Org. Chem. Vol. 73, No. 10, 2008 3723

Plant et al.
(MgSO4), and evaporated. Flash chromatography (1:3 hexane/
EtOAc) gave a white solid (2.34 g, 5.84 mmol, 91%). HRMS (ESI)
m/z calcd for C16H8N2O3F5S [M + H]+ 403.0176, found 403.0159.
1H NMR (250 MHz, CDCl3): δ 2.60 (s, 3H), 7.53 (dd, J ) 7.9 Hz,
4.9 Hz, 1H), 8.29 (ddd, J ) 7.9 Hz, 2.1 Hz, 1.5 Hz, 1H), 8.72 (dd,
J ) 5.2 Hz, 1.5 Hz, 1H), 9.07 (d, J ) 2.1 Hz, 1H). 13C NMR (60
MHz, CDCl3): δ 13.8, 106.7, 123.4, 124.2, 132.6, 136.3, 138.0,
139.2, 140.0, 143.3, 148.4, 151.1, 156.7, 163.1, 172.3.
dissolved in MeOH (2 mL) and triethylamine (0.12 mL, 0.87 mmol)
was added. The reaction was stirred for 24 h at room temperature
under Ar. The solution was then diluted with water (2 mL) and
acidified to pH 4. Solvents were then removed under vacuum and
the remaining yellow solid was purified by flash chromatography
(EtOAc to 3:1 EtOAc/MeOH) to give a white solid (111 mg, 0.21
mmol, 74%). HRMS (ESI) m/z calcd for C21H21N5O9S.Na [M +
Na]+ 542.0958, found 542.0938. 1H NMR (250 MHz, DMSO-d6):
δ 3.60 (s, 3H), 3.93-4.14 (m, 5H), 4.71 (dd, J ) 7.9 Hz, 5.8 Hz,
1H), 5.34 (d, J ) 4.3 Hz, 1H), 5.48 (d, J ) 5.7 Hz, 1H), 5.57 (d,
J ) 7.9 Hz, 1H), 5.78 (d, J ) 6.7 Hz, 1H), 7.07 (dd, J ) 6.5 Hz,
4.9 Hz, 1H), 7.32 (d, J ) 7.9 Hz, 1H), 7.51 (d, J ) 8.2 Hz, 1H),
7.61 (t, J ) 7.3 Hz, 1H), 8.39 (d, J ) 4.3 Hz, 1H), 9.86 (d, J ) 8.2
Hz, 1H), 11.35 (bs, 1H). 13C NMR (60 MHz, MeOD-d4): δ 37.54,
51.5, C5′, 69.7, 73.0, 73.2, 84.1, 88.3, 101.8, 102.1, 119.5, 121.5,
136.5, 140.6, 148.8, 151.0, 159.0, 164.4, 169.5, 170.3, 186.9.
Methyl-1,5-dideoxy-1-(3,4-dihydro-2,4-dioxo-1-(2H)-pyrim-
idinyl)-5-[5-(pyridin-3-yl)isoxazole-4-carbonylamino]- ꢀ-D-allo-
furanuronate (33). UPOC methyl ester 4a (647 mg, 2.15 mmol)
and isoxazole 32 (1.30 g, 3.23 mmol) were dissolved in dry DMF
(5 mL) and stirred at room temperature under Ar. After 48 h the
solvent was removed, and the residue was dissolved in water (30
mL) and extracted with EtOAc. The combined organic layers were
dried (MgSO4) and evaporated. Flash chromatography (EtOAc to
4:1 EtOAc/MeOH) gave a cream-colored solid (286 mg, 0.55 mmol,
26% from compound 3a). HRMS (ESI) m/z calcd for
Acknowledgment. We are grateful to Eric Clarke (physico-
chemical studies, Syngenta), Beth Shirley (physicochemical
studies, Syngenta), Kate Sharples (metabolism studies, Syn-
genta), and Victoria Glancy (metabolism studies, Syngenta) and
thank the BBSRC and Syngenta for funding.
1
C21H21N5O9NaS [M + Na]+ 542.0958, found 542.0933. H NMR
(250 MHz, DMSO-d6): δ 2.57 (s, 3H), 3.66 (s, 3H), 3.96-4.00
(m, 1H), 4.07-4.20 (m, 2H), 4.85 (t, J ) 7.0 Hz, 1H), 5.32 (d, J
) 5.5 Hz, 1H), 5.54 (d, J ) 5.5 Hz, 1H), 5.64 (d, J ) 7.9 Hz, 1H),
5.75 (d, J ) 5.8 Hz, 1H), 7.55-7.59 (m, 2H), 8.13-8.17 (m, 1H),
8.71 (d, J ) 4.0 Hz, 1H), 8.92 (bs, 1H), 9.23 (d, J ) 7.6 Hz, 1H),
11.39 (bs, 1H). 13C NMR (125 MHz, DMSO-d6): δ 13.3, 52.1, 54.0,
70.1, 71.5, 82.2, 88.4, 102.2, 112.1, 122.5, 124.0, 134.7, 141.2,
147.6, 150.7, 151.6, 160.4, 160.4, 162.9, 164.8, 169.0.
Supporting Information Available: Experimental details for
1
compounds 2b, 3b, 4b, 5, 6a, 6b, 27, 28, 31, 34; H and 13C
NMR spectra for all novel compounds described in the
Experimental Section. This material is available free of charge
via the Internet at http://pubs.acs.org.
Methyl-1,5-dideoxy-1-(3,4-dihydro-2,4-dioxo-1-(2H)-pyrim-
idinyl)-5-[2-cyano-3-oxo-4-(pyridine-2-ylthio)butanoylamino]-ꢀ-
D-allofuranuronate (35). Nucleoside 33 (150 mg, 0.29 mmol) was
JO702564Y
3724 J. Org. Chem. Vol. 73, No. 10, 2008