Ring-modified analogues and molecular dynamics studies to probe the requirements for fungicidal activities of malayamycin A and its N-nucleoside variants

Article abstract of DOI:10.1021/jo070520d

(Chemical Equation Presented) The importance of functional group orientations and the integrity of the bicyclic perhydrofuran core of malayamycin A and two equally active N-nucleoside analogues as fungicides were investigated. Two analogues 10 and 11, representing a THP-truncated and a bicyclic aza-variant, were synthesized and found to be inactive. Molecular dynamics studies on malayamycin A and analogues were performed to highlight the importance of properly orientating the urea and methyl ether groups.

Full text of DOI:10.1021/jo070520d

Ring-Modified Analogues and Molecular Dynamics Studies To
Probe the Requirements for Fungicidal Activities of Malayamycin A
and Its N-Nucleoside Variants
Olivier Loiseleur,*,‡ Dougal Ritson,† Mafalda Nina,‡ Patrick Crowley, Trixie Wagner,§ and
Stephen Hanessian*,†
Syngenta, Crop Protection Research, Schwarzwaldallee 215, CH-4002 Basel, Switzerland, Syngenta,
Jealott’s Hill International Research Centre, Bracknell, Berkshire, RG45 6EY, United Kingdom,
Department of Chemistry, UniVersite´ de Montre´al, C. P. 6128, Succ. Centre-Ville, Montre´al, P.Q., Canada,
H3C 3J7, NoVartis Institutes for Biomedical Research, NoVartis Pharma AG, Lichtstrasse 35, CH-4056
Basel, Switzerland
oliVier.loiseleur@syngenta.com; stephen.hanessian@umontreal.ca
ReceiVed March 13, 2007
The importance of functional group orientations and the integrity of the bicyclic perhydrofuran core of
malayamycin A and two equally active N-nucleoside analogues as fungicides were investigated. Two
analogues 10 and 11, representing a THP-truncated and a bicyclic aza-variant, were synthesized and
found to be inactive. Molecular dynamics studies on malayamycin A and analogues were performed to
highlight the importance of properly orientating the urea and methyl ether groups.
Introduction
A3 3 and ezomycin A24 4 are representative of such bicyclic
N-nucleosides, while ezomycin B15 5 is a C-nucleoside equiva-
lent (Figure 1). Malayamycin A is the most recent entry in this
group. In line with the ezomycins that have been reported to
exhibit antifungal and antibacterial activities,6 malayamycin A
displays broad spectrum activity against phytopathogenic fungi
in the greenhouse,7 with a yet unknown mode of action. In
contrast to the ezomycins, malayamycin A has a lower molecular
Malayamycin A (1, Figure 1) is a naturally occurring
C-nucleoside discovered at Syngenta Crop Protection Labora-
tories. It was isolated from the soil organism Streptomyces
malaysiensis, and its structure was determined by extensive
NMR studies and degradative work.1 Further production of
malayamycin A by fermentation delivered the novel desmethyl
malayamycin A (2), which was isolated as a minor metabolite.2
Within the class of nucleosides, only a handful of naturally
occurring N- and C-pyrimidine nucleosides feature a bicyclic
perhydrofuran motif rather than the commonly encountered
monocyclic pentofuranosyl or hexofuranosyl core. Octosyl acid
(3) Isono, K.; Crain, P.-F.; McCloskey, J. A. J. Am. Chem. Soc. 1975,
97, 943. For the total synthesis of octosyl acid A, see: (a) Knapp, S.; Thakur,
V. V.; Madurru, M. R.; Malolanarasimhan, K.; Morriello, G. J.; Doss, G.
A. Org. Lett. 2006, 8, 1335. (b) Danishefsky, S.; Hungate, R. J. Am. Chem.
Soc. 1986, 108, 2486. (c) Hanessian, S.; Kloss, J.; Sugawara, T. J. Am.
Chem. Soc. 1986, 108, 2758.
(4) (a) Sakata, K.; Sakurai, A.; Tamura, S. Agric. Biol. Chem. 1975, 39,
885. (b) Sakata, K.; Sakurai, A.; Tamura, S. Tetrahedron Lett. 1974, 49,
4327. (c) Sakata, K.; Sakurai, A.; Tamura, S. Agric. Biol. Chem. 1973, 37,
697.
(5) Sakata, K.; Sakurai, A.; Tamura, S. Tetrahedron Lett. 1975, 16, 3191.
(6) (a) Sakata, K.; Sakurai, A.; Tamura, S. Agric. Biol. Chem. 1977, 41,
2027. (b) Sakata, K.; Sakurai, A.; Tamura, S. Agric. Biol. Chem. 1974, 38,
1883.
* Address correspondence to this author.
‡ Syngenta, Crop Protection Research & Technology.
Syngenta, Jealott’s Hill International Research Centre.
† Universite´ de Montre´al.
§ Novartis Institutes for Biomedical Research, Novartis Pharma AG.
(1) Benner, J. P.; Boehlendorf, B. G. H.; Kipps, M. R.; Lambert, N. E.
P.; Luck, R.; Molleyres, L.-P.; Neff, S.; Schuez, T.-C.; Stanley, P. D. Patent
appl. No. WO2003062242, July 31, 2003.
(2) Neff, S.; Bo¨hlendorf, B.; Winkler, T. Unpublished results.
10.1021/jo070520d CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/21/2007
J. Org. Chem. 2007, 72, 6353-6363
6353

Loiseleur et al.
FIGURE 2. Ball-and-stick models (PLATON) of malayamycin A
hydrate (1) and desmethyl malayamycin A hydrate (2) based on X-ray
data. The structure-based designed tricyclic 1-cytosinyl- N-malayamy-
cin, 9.
modification at this site resulted in loss of fungicidal activity.9a
We next turned our attention to the three-dimensional orientation
of the urea onto the perhydrofuropyran scaffold. In the course
of our synthetic efforts we obtained X-ray quality crystals of 1
and 2 from mixtures of ethanol/water (1) and isopropanol/water
(2) after slow evaporation.10 The three-dimensional crystal
structures as depicted in Figure 2 revealed several interesting
features. The trans-fused furanoside in the perhydrofuropyran
bicyclic system is locked in the C3-endo puckering mode.
Accordingly, the C5′-substituted pyrimidinone adopts an anti
(7) Malayamycin A 1 and all the analogues were tested against three
foliar fungal diseases of plants. The compounds were diluted in reverse
osmosis water to a final concentration of 100 ppm in water (1 mg of
compound in a final volume of 10 mL) immediately before use. TWEEN
20 (registered trade mark, at a final concentration of 0.05% by volume)
was added with the water to improve retention of the spray deposit. The
compounds were applied to the foliage of the test plants grown on an
artificial, cellulose based growing medium, by spraying the plant to
maximum droplet retention. Tests were carried out against Stagonospora
Nodorum (LEPTNO), Blumeria graminis f.sp. tritici (ERYSGT), and
Puccinia triticina (PUCCRT) on wheat. Two replicates, each containing
three plants, were used for each treatment. The plants were inoculated with
either a calibrated fungal spore suspension or a “dusting” with dry spores
6 h (ERYSGT) or 1 day (PUCCRT and LEPTNO) after chemical
application. The plants were then incubated under high humidity conditions
(except those inoculated with Blumeria graminis f.sp. tritici) and put into
an appropriate environment to allow infection to proceed until the disease
was ready for assessment. The time period between chemical application
and assessment varied from six to nine days according to the disease and
environment. However, each individual disease was assessed after the same
time period. The level of disease present (the percentage leaf area covered
by actively sporulating disease) was assessed visually and the assessed values
for all replicates were averaged to provide mean disease values. For
malayamycin A 1 and 1-cytosinyl-N-malayamycin A 7 there was 0% fungal
infection present for all three fungal pathogens, indicating 100% control.
Desmethyl malayamycin A 2 gave 100% control of LEPTNO and PUCCRT,
but 0% control of ERYSGT. The C6-methoxy epimer 42 gave 70% control
of PUCCRT and 0% of LEPTNO and ERYSGT. All the other analogues
tested gave 0% control of all three diseases.
FIGURE 1. Structures of N- and C-nucleosides with a bicyclic dioxa
perhydrofuropyran motif.
weight and does not contain carboxylic acid functions, which
makes it a more appropriate lead for crop protection. Recently,
the proposed structure and absolute stereochemistry of 1 were
confirmed by a stereocontrolled total synthesis.8 By analogy
with the naturally occurring N- and C-ezomycins, N-malaya-
mycin A (6) and the related pyrimidine and purine congeners
(7 and 8, Figure 1) were synthesized via a synthetic route
allowing for anomeric diversity.9 Biological screening revealed
the 1-cytosinyl-N-nucleoside 7 to be equivalent to malayamycin
A in terms of fungicidal activity. On the other hand, related
pyrimidine and purine congeners including N-malayamycin A
(6) and 9-adenyl-N-malayamycin A (8) were either weak or
completely inactive.
(8) Hanessian, S.; Marcotte, S.; Machaalani, R.; Huang, G. Org. Lett.
2003, 5, 4277.
Results and Discussion
(9) (a) Hanessian, S.; Marcotte, S.; Machaalani, R.; Huang, G.; Pierron,
J.; Loiseleur, O. Tetrahedron 2006, 62, 5201. (b) Hanessian, S.; Machaalani,
R.; Marcotte, S. Patent appl. No. WO 2004069842, August 19, 2004. (c)
Loiseleur, O.; Schneider, H.; Huang, G. H.; Machaalani, R.; Selles, P.;
Crowley, P.; Hanessian, S. Org. Proc. Res. DeV. 2006, 10, 518. (d)
Hanessian, S.; Huang, G.; Chenel, C.; Machaalani, R.; Loiseleur, O. J. Org.
Chem. 2005, 70, 6721. (e) Hanessian, S.; Marcotte, S.; Huang, G.; Crowley,
P. J.; Loiseleur, O. Patent appl. No. WO 2005005432, January 20, 2005.
With this knowledge in hand, it was deemed appropriate to
gain some information regarding the importance for biological
activity of the functional groups displayed on the tetrahydro-
pyran ring in 7. The urea group, which is commonly shared
with the ezomycins, proved to be required as functional group
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Requirements for Fungicidal ActiVities
SCHEME 1. Synthesis of Compound 10
FIGURE 3. Truncated (10) and constrained (11) analogues of 7.
orientation to the furanoside ring, with the C2′-carbonyl group
pointing away from the tetrahydrofuran ring oxygen.11 The urea
group adopts an orthogonal position, which bisects the plane
of the chairlike tetrahydropyran ring of the bicyclic system with
the carbonyl group oriented “outward”. We utilized the three-
dimensional functional characteristics shown in the crystal
structures of 1 and 2 to derive the tricyclic N-cytosinyl analogue
9, in which the axially orientated urea group was connected to
the perhydrofuropyran core by an ethano bridge (Figure 2).
Preliminary superposition of a modeled structure optimized
in vacuo with a semiempirical method over the X-ray structure
of 1 showed excellent congruence with the urea orientation being
locked by the ethano bridge. Accordingly, a synthetic route to
9 was developed12 although biological testing showed 9 to be
devoid of fungicidal activity. To further probe the orientations
of the urea and methoxy groups in relation to the bicyclic core,
we embarked on the synthesis of the monocyclic and bicyclic
N-nucleosides 10 and 11 (Figure 3). In 10 we “relaxed” the
urea and the adjacent C6-methoxy groups by allowing them to
adopt freely rotating preferred orientations in a truncated
tetrahydropyran. Compound 11 represents a constrained ana-
logue with a fixed urea orientation. The methoxy oxygen in 11
was intended to mimic the tetrahydropyran oxygen, possibly a
H-bond acceptor.
Diacetone-D-glucose 12 was an appropriate starting material,
possessing functional handles in all the relevant positions when
compared to the target 10 (Scheme 1). Oxidation of 12 and
reduction of the corresponding ketone with NaBH4 in MeOH
gave the known alcohol 1313 in 75% yield. The epimer (not
shown) was recovered in 15% yield and as oxidation of 12 was
essentially quantitative, the stereoselectivity of the hydride
addition was reflected in the yields of the epimers. O-
Methylation of 13 was performed by using NaH and MeI in
THF and the resulting methyl ether was stirred in 80% AcOH
in H2O for 48 h to hydrolyze the distal acetonide. Attempts to
selectively methylate the primary alcohol of 14 with the
Meerwein salt (Me3O+BF4- 14 were unsuccessful, so the primary
)
alcohol was protected as the tert-butyldiphenylsilyl ether 15.
Next, we had to install an azide group at C5, later to be converted
to the urea in 10, but the secondary alcohol of 15 did not have
the requisite stereochemistry. Hence, a double inversion was
required and the first displacement was carried out under
Mitsunobu conditions, using chloroacetic acid as the nucleophilic
component.15 Although the reaction was slow, the desired ester
was recovered in reasonable yield after 4 days, and was then
subjected to saponification with K2CO3 in MeOH to provide
the inverted secondary alcohol 16. The second inversion, and
introduction of the azide group, was performed on 16 under
Mitsunobu conditions in the presence of diphenyl phosphoryl
azide in THF. Compared to 15, the reaction was remarkably
faster, giving the azide 17 in high yield after 18 h. Liberation
of the primary alcohol of 17 with use of TBAF in THF gave
(10) Compounds 1 and 2 both show a modulated structure (see: Chapuis,
G.; Scho¨nleber A. Chimia 2001, 55, 523). To obtain coordinates suitable
as starting model for the molecular dynamics simulations the modulated
structures were approximated by the corresponding superstructures which
revealed eight virtually identical molecular conformations for compound 1
and four for compound 2. Coordinates of those approximations will not be
deposited as the modulated structure of compound 1 will be described in
detail in a forthcoming paper (see also: Loiseleur, O.; Wagner, T.;
Scho¨nleber, A.; Petricek, V. Deutsche Gesellschaft fu¨r Kristallographie
(DGK) 15. Jahrestagung, 2007 Bremen, conference abstracts).
(11) Saenger W. In The Principles of Nucleic Acid Structure; Springer,
Verlag: New York, 1983.
(12) Hanessian, S.; Ritson, D. J. J. Org. Chem. 2006, 71, 9807.
(13) Christensen, S. M.; Hansen, H. F.; Koch, T. Org. Process Res. DeV.
2004, 44, 777.
(14) Meerwein, H.; Hinz, G.; Hofmann, P.; Kroning, E.; Pfeil, E. J. Prakt.
Chem. 1937, 147, 257.
(15) Martin, S. F.; Dodge, J. A. Tetrahedron Lett. 1991, 32, 3017.
J. Org. Chem, Vol. 72, No. 17, 2007 6355

Loiseleur et al.
SCHEME 2. Synthesis of Compound 11
18 as a colorless, crystalline solid from which single-crystal
X-ray analysis could be performed, thereby confirming that the
sequence of inversions had occurred as intended.16 O-Methy-
lation of the alcohol in 18 was carried out under standard
conditions to give 19, which then had to be manipulated so that
the anomeric position could be readily activated during the
nucleobase coupling step. For this purpose thioglycosides have
been used in conjunction with thiophilic reagents to good
effect.9a,c,d,17 The acetonide 19 was stirred with thiophenol and
Amberlyst-15 in CH2Cl29d,12 leading to a very low yield (10%)
of the thioglycoside 20 in addition to several other reaction
products. Since other methods such as PhSH/BF3‚OEt2, PhSH/
TFA, and PhS-TMS/ZnI218 gave similar results, we then turned
to a Vorbru¨ggen coupling.19 Hence, 19 was stirred in 50%
aqueous TFA for 2 h and the corresponding diol was acetylated
to give 21 as a 1:1 mixture of anomeric acetates. Subsequent
treatment with 4-(N-trimethylsilyl)acetamido-2-(trimethylsilyloxy)-
pyrimidine17a,b in the presence of SnCl4 in dichloroethane gave
the N-nucleoside 22 in good yield. Finally, reduction of the azide
group under Staudinger conditions,20 urea formation, and final
deprotection9d,12 gave the desired compound 10. The determi-
nation of the stereochemistry at the anomeric position was
secured by NOE experiments. Irradiation of the cytosinyl proton
C6′-Ha gave a good enhancement (3.4%) of the proton C3-Hb to
confirm that the nucleobase was in the â-position (Scheme 1).
The synthesis of 11 commenced with the known alcohol 23,21
which was methylated, and the desired diol 24 was unmasked
by stirring in 80% AcOH/H2O (Scheme 2). Oxidative cleavage
of the vicinal diol was performed with sodium periodate to give
the corresponding aldehyde 25 in high yield. Attempts to form
the bis-aldehyde 26 that was to be utilized in a double reductive
amination sequence to form the piperidine 27, with NaIO4 and
catalytic OsO4, resulted in the trapping of the intermediate diol
as the hemiacetal 28. Since ozonolysis of 25 was also unsuc-
cessful, a second approach was investigated where the nitrogen,
to be incorporated into the piperidine ring of 11, would be
installed first followed by azabicycle formation. We first
considered an intramolecular “aza-Wittig” reaction. Hence, 29
was subjected to standard ozonolysis conditions followed by
treatment with NaBH4 to give 30. Although a Mitsunobu
reaction (DPPA, PPh3, DIAD, THF) of the primary alcohol of
30 gave the corresponding primary azide 31 in 77% yield, we
chose the higher yielding 2-step sequence of mesylation with
subsequent azide displacement (86% over 2 steps). Again,
aqueous AcOH was employed to remove the acetonide providing
the vicinal diol 32, which was subsequently cleaved with sodium
periodate to give the aldehyde 33. Although formation of the
“aza-ylide” with PPh3 in Et2O and cyclization were successful,
the resulting imine 34 underwent partial epimerization at C7,
possibly due to formation of the enamine intermediate 35. An
alternative approach based on an intramolecular SN2 cyclization
is shown in Scheme 3. Thus, the aldehyde 33 was reduced with
NaBH4 and the primary alcohol was activated as the mesylate
36. Upon catalytic hydrogenation of the azide group with Pd
black in MeOH/Et3N, facile ring closure took place to afford
the expected bicyclic oxapiperidine. We had found in earlier
studies that the robust tert-butylsulfonyl22 (Bus) protecting group
for amines could be removed under conditions compatible with
N-nucleosides.12 Consequently, the intermediate bicyclic amine
was treated with tert-butylsulfinyl chloride, and the resulting
tert-butylsulfinyl amide was oxidized with m-CPBA to the tert-
butylsulfonyl amide 37. Treatment of 37 with PhSH/Amberlyst-
15 in CH2Cl2 gave the thioglycoside 38 directly in a 5:1 ratio
of R:â anomers, respectively, rather than the expected diphenyl
dithioacetal that had previously been observed in this series,9a,d,12
albeit in modest yield. Protection of the alcohol as the pivaloyl
ester and treatment with NIS/TfOH and bis-silylated cytosine
gave the expected nucleoside 40 in 76% yield. The â-stereo-
chemistry at the anomeric position was determined by 1H NMR
(5.95 ppm, J ) 0 Hz). Removal of the Bus group of 40 was
(16) See the Supporting Information, S46.
(17) (a) Knapp, S.; Shieh, W-C.; Jaramillo, C.; Triller, R.; Nandau, S.
R. J. Org. Chem. 1994, 59, 946. (b) Knapp, S.; Shieh, W-C. Tetrahedron
Lett. 1991, 32, 3627. (c) Hanessian, S.; Sato, K.; Liak, T. J.; Danh, N.;
Dixit, D.; Cheney, B. V. J. Am. Chem. Soc. 1984, 106, 6114. (d) Hanessian,
S.; Dixit, D. M.; Liak, T. J. Pure Appl. Chem. 1981, 53, 129.
(18) Hanessian, S.; Guindon, Y. Carbohydr. Res. 1980, 86, C3-C6.
(19) Vorbru¨ggen, H.; Ho¨fle, G. Chem. Ber. 1981, 114, 1256.
(20) (a) Vaultier, M.; Knouz, N.; Carrie, R. Tetrahedron Lett. 1983, 24,
763. (b) Staudinger, H.; Meyer, J. HelV. Chim. Acta 1919, 2, 635.
(21) Banerjee, S.; Ghosh, S. J. Org. Chem. 2003, 68, 3981.
22,23
achieved with TfOH in the presence of anisole in CH2Cl2
(22) Sun, P.; Weinreb, S. M. J. Org. Chem. 1997, 62, 8604.
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Requirements for Fungicidal ActiVities
SCHEME 3. Synthesis of Compound 11
FIGURE 4. C6 Analogues of 1-cytosinyl-N-malayamycin A (7).
ligands do not bind to proteins in their global energy minimum
conformation in vacuo, many pharmacophore models have been
based on such models in cases where no additional information
is available.24 The X-ray structure of 1 was used as a starting
conformation to predict the conformational preferences of 7 in
vacuo with use of MMFF, an empirical molecular mechanics
method. Starting conformations for congeners 42 and 43 were
derived from the X-ray structure and modified as appropriate.
The Monte Carlo conformational analysis search technique was
used to locate the conformational distribution of the active
1-cytosinyl-N-malayamycin A 7, the inactive C6-fluoro-N-
malayamycin 43, and the epi-C6-methoxy-N-malayamycin 42.
The predicted global minimum and two local minima for each
analogue were subsequently geometry optimized with the HF/
3-21G* ab initio method (Figure 5).
Comparison of the crystal structure of malayamycin A with
the global energy minimum conformation of 7 in vacuo shows
that major topological features such as the dihedral angle for
the pyrimidinone and the urea groups are mostly conserved,
and that the tetrahydropyran ring is in a chair conformation (see
Figure 5A). Not surprisingly, the rotational position of the
pyrimidinone is well in line with that found in nucleotides, i.e.,
the base is anti with respect to the furanoside ring.11 In the
calculated global minimum, both the C6-OMe and the O1 atom
are equatorially disposed in a symmetric fashion relative to the
urea and the urea-NH interacts with C6-OMe through an
intramolecular hydrogen bond. On the other hand, the next local
energy minimum corresponds to a conformation displaying an
intramolecular hydrogen bond between the urea-NH, O1 and
the C2′-carbonyl group of the now syn-oriented pyrimidinone.
This energy minimum and the global minimum are almost iso-
energetic. Accordingly, the related conformations should be
equally available.
When C6-OMe is removed from its position adjacent to the
urea, as in the inactive C6-fluoro-1-cytosinyl-N-malayamycin
A (43) and the weakly active epi-C6-methoxy-N-malayamycin
42, this equilibrium is disrupted. Without the methoxy in
proximity to the urea, the intramolecular hydrogen bond
interaction of O1 and the pyrimidinone with the urea now
predominates and the base is locked in a syn orientation. For
43, the lowest energy geometries in vacuo correspond to a
tetrahydropyran ring in a twist boat and a chair conformation
(see Figure 5B). For 42, the lowest energy geometry corresponds
to the chair conformation with an intramolecular H-bond of the
urea-NH with the pyrimidinone C2′-carbonyl (see Figure 5C).
Thus, it appears that the derivatives 42 and 43 do not allow the
urea group and the pyrimidinone to be oriented as in 1-cytosinyl-
and the final sequence of urea installation and deprotection was
carried out as before to give 11 in good yield. Not unexpectedly,
the structurally simplified analogues 10 and 11 were also devoid
of activity, joining a long list of other chemically modified
congeners relegated to the ranks of inactive fungicides.9a,d,12
Clearly the unique spatial arrangement of the urea and methoxy
groups, in conjunction with the topology of the dioxabicyclic
core structure in malayamycin or its N-nucleoside analogues 6
and 7, are critical features for containing their potent fungicidal
activity.
We then decided to study the preferred conformations of
malayamycin A and congeners in vacuo using an ab initio RHF/
3-21G* method and in water using molecular dynamics simula-
tions. We chose the C6-methoxy substituent as an entry point.
To understand the functional importance of the methyl ether,
we considered desmethyl malayamycin A 2 and a series of C6-
modified 1-cytosinyl-N-malayamycin analogues which had been
prepared previously (Figure 4).9a,d Although not as active as
malayamycin A 1, desmethyl malayamycin A 2 had significant
fungicidal activity, whereas the more bulky ethoxy-substituted
congener 41 was inactive. The C6-methoxy epimer 42 had much
weaker fungicidal activity and the C6-fluoro-substituted analogue
43 was inactive. This structure-activity pattern points to the
possible contribution of an intermolecular hydrogen bond to C6-
OMe in the binding of malayamycin A at the active site.
However, other effects might be at work.
At that stage, we probed the conformational features of
1-cytosinyl-N-malayamycin A (7) and derivatives 42 and 43,
and compared preferred conformations in vacuo and in water
to develop a hypothesis for the optimal conformation required
for activity. Although there is considerable evidence that many
(24) (a) Nicklaus, M. C.; Wang, S.; Driscoll, J.; Milne, G. W. A. Bioorg.
Med. Chem. 1995, 3, 411. (b) Vieth, M.; Hirst, J. D.; Brooks, C. L., 3rd J.
Comput.-Aided Mol. Des. 1998, 12, 563. (c) Bostro¨m, J.; Norrby, P.-O.;
Liljefors, T. J. Comput.-Aided Mol. Des. 1998, 12, 383. (d) Debnath, A. K.
J. Med. Chem. 1999, 42, 249. (e) Perola, E.; Charifson, P. S. J. Med. Chem.
2004, 47, 2499.
(23) Hanessian, S.; Del Valle, J. R.; Xue, Y.; Blomberg, N. J. Am. Chem.
Soc. 2006, 128, 10491.
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Loiseleur et al.
FIGURE 5. Conformational analysis of 7 (A), 43 (B), and 42 (C) in vacuo. ∆E is the internal energy difference between the global energy
minimum and the local energy minimum geometries optimized with the RHF/3-21G* method.
N-malayamycin A 7. Therefore, it is hypothesized that the
orientation of the urea group and the pyrimidinone is important
for activity and that this orientation is influenced by the C6-
methoxy group.
The barrier of rotation of the urea dihedral angle C7a-C6-
N-C(O) of 1-cytosinyl-N-malayamycin A (7) was assessed by
an adiabatic dihedral driving calculation with the HF/3-21G*
ab initio molecular orbital method (Figure 6). Figure 6 shows
6358 J. Org. Chem., Vol. 72, No. 17, 2007

Requirements for Fungicidal ActiVities
FIGURE 7. Structure of 44 in the crystal. Atomic displacement
ellipsoids drawn at the 50% probability level, hydrogen atoms drawn
as spheres of arbitrary radius (PLATON).
tetrahydropyran unit and the C6-OMe delineates their importance
for the biological activity of malayamycins.
Conclusion
FIGURE 6. Ab initio HF/3-21G* torsional potential for rotation around
the urea side chain of 1-cytosinyl-N-malayamycin A (7).
In the course of our studies we have shown the importance
of the substituent orientations and stereochemistry in a malaya-
mycin-type core structure for maintaining fungicidal activity.
The hypothesis that emerges from these studies is that in the
topologically unique malayamycin core structure, a highly
organized orientation of substituents is necessary for activity.
In this regard, it is still noteworthy that activity was maintained
in N-malayamycin and the corresponding N-cytosinyl ana-
logues.9c-e
two key structures from the torsional curve for geometry
optimizations with fixed urea dihedral angles. The two confor-
mations were found equally energetic at this level of calculation,
i.e., with the urea side chain oriented at 82° (similar to the X-ray
structure) and at -103° (hydrogen bond between C6-OMe and
the urea-NH). Moreover, the torsional curve indicates that over
the range from 0 to 120°, the energy differs by only 2
kcal‚mol-1. On the other hand, the calculations suggest that both
equally energetic conformations are separated by 18 kcal‚mol-1
free energies of activation and hence may not easily interconvert
at room temperature.
Experimental Section
1,2:5,6-Di-O-isopropylidene-D-allofuranose (13). To a dry flask
was added CrO3 (3.91 g, 39.1 mmol), under Ar atmosphere,
containing anhydrous CH2Cl2 (70 mL), which was cooled to 0 °C.
Subsequently molecular dynamics simulations (MD) in water
were performed on derivatives 7, 42, and 43 in order to predict
the solvation effect on the sampled population of conformers
and the urea side chain orientation, using the protocol de-
scribed.27 One major population for 1-cytosinyl-N-malayamycin
A (7) accounting for 30% of the sampled conformations was
observed during the MD simulation with an average conforma-
tion close to the lowest energy conformer in vacuo and an
average urea dihedral angle C7a-C6-N-C(O) of 85 ( 3°. For
the epi-C6-methoxy-N-malayamycin (42), the predominant
population (52%) had an average urea dihedral angle of 148 (
26°. For the C6-fluoro-1-cytosinyl-N-malayamycin A (43), two
major populations representing 30% and 27% of the overall
population were sampled within 20 ns of MD with average
dihedral angles of 127 ( 22° and 85 ( 3°, respectively. Overall
the MD simulations analysis of the conformational space
revealed the influence of the C6-OMe on the orientation of the
urea side chain.
To probe the influence of the oxygen atom C3a-O-C5 in
the tetrahydropyran ring in N-malayamycin 7, we prepared the
N-cytosinyl carba-analogue 44 (Figure 7).9a X-ray quality
crystals of 44 were obtained by slow evaporation from isopro-
panol, and the three-dimensional solid-state structure revealed
no conformational differences compared to malayamycin A.
Surprisingly, analogue 44 was devoid of fungicidal activity in
spite of its virtually identical topology compared to the oxa-
analogue 7, which is equipotent to malayamycin A 1. The poor
tolerance to modifications of the oxygen incorporated in the
(27) Molecular dynamics simulations in explicit water were performed
on 1-cytosinyl-N-malayamycin A (7) and congeners 42 and 43 with the
molecular mechanics CHARMm software and CHARMM22 force field
(Accelrys). The MD simulation used the continuum reaction field treatment
of long-range SSBP module (Beglov; Roux J. Chem. Phys. 1994, 100, 9050)
as implemented in the CHARMm module from InsightII (Accelrys). Force
field parameters for the ligands consistent with the CHARMM22 force field
were produced with the software WitNotP (Novartis Pharma). Three
independent MD simulations were performed with the crystal structure of
1-cytosinyl-N-malayamycin A (7), and global minimum optimized geom-
etries HF/3-21G* for 42 and 43 as starting conformations. A spherical region
of 14 Å radius was simulated in atomic detail, including the inhibitor and
350 TIP3 water molecules for a total of 1101 atoms. This sphere size ensured
that the solute has a solvation shell of explicit water at least 8 Å thick in
all directions. The solvent outside this sphere was treated as a dielectric
continuum with a dielectric constant of 80. The inner region has a dielectric
of 1. Atomic partial charges in the inner region polarize the outer continuum,
giving rise to a reaction field on each explicit atom, which is approximated
here by a spherical harmonic expansion of order 15. Within the spherical
region, electrostatic interactions were treated without any truncation by using
a multipole approximation. Each simulation was run for 20 ns and
trajectories were analyzed with an in-house clustering method derived from
a previous work (Hamprecht, F. A.; Peter, C.; Daura, X.; Thiel, W.; van
Gunsteren, W. F. J. Chem. Phys. 2001, 114, 2079). For each simulation,
10 000 structures were extracted from the last 10 ns of the trajectory at 1
ps intervals for analysis. The clustering was performed in Cartesian space.
For each structure, a least-square translational and rotational fit was
performed with the heavy atoms of residues 4-11 and the atom positional
root-mean-square difference (RMSd) for this set of atoms was calculated.
Terminal residues were not taken into account, as they tend to have more
freedom of motion. The number of structures satisfying the similarity
criterion set to RMSd e1 Å for the heavy atoms was determined in the
pool of 10 000 structures. The structure with the highest number of neighbors
(i.e. structures satisfying the similarity criterion) was taken as the central
member of the first cluster. All structures belonging to the first cluster were
removed from the pool. The number of neighbors was computed again with
the remaining structures. The structure with the highest number of neighbors
becomes the central member of the second cluster. The process is iterated
until all structures were assigned to a cluster.
(25) Kapur, B. M.; Allgeier, H. HelV. Chim. Acta 1968, 51, 89.
(26) Lee, J.-C.; Chang, S.-W.; Liao, C.-C.; Chi, F.-C.; Chen, C.-S.; Wen,
Y.-S.; Wang, C.-C.; Kulkarni, S.-S.; Puranik, R.; Liu, Y.-H.; Hung, S.-C.
Chem. Eur. J. 2004, 10, 399.
J. Org. Chem, Vol. 72, No. 17, 2007 6359

Loiseleur et al.
Anhydrous pyridine (11.8 mL, 11.5 g, 146 mmol) and Ac2O (7.0
mL, 7.56 g, 74.0 mmol) were added followed by diacetone-D-
glucose (6.00 g, 23.1 mmol), which was added portionwise over
10 min. After being stirred for 30 min the mixture was brought to
room temperature and stirred a further 2 h. The black solution was
poured into EtOAc (ca. 150 mL) and the mixture was filtered
through silica washing with EtOAc. The solvents were removed
and the residue pumped overnight.
127.36, 112.6, 103.6, 80.0, 77.2, 71.5, 64.0, 57.6, 26.5, 26.4, 26.2,
18.9; HRMS (ESI) calcd for C26H36O6SiNa [M + Na] 495.2173,
found 495.2194.
6-O-tert-Butyldiphenylsilyl-1,2-O-isopropylidene-3-O-methyl-
L-talofuranose (16). To a solution of 15 (1.44 g, 3.05 mmol) in
dry toluene (38 mL), under Ar atmosphere, was added PPh3 (1.61
g, 6.14 mmol) and chloroacetic acid (720 mg, 7.62 mmol), after
which the solution was cooled to 0 °C. DIAD (1.20 mL, 1.23 g,
6.08 mmol) was added dropwise to the solution and the mixture
was allowed to warm to room temperature. After 6 days the solvents
were evaporated under vacuum and the product was purified by
flash chromatography (hexanes-EtOAc, 4:1) to give the desired
product. However, the material could not be obtained in a pure
form so it was used in the next step.
The crude oil was dissolved in MeOH (20 mL) and NaBH4 (1.30
g, 34.4 mmol) was added portionwise. After 3 h MeOH (50 mL)
was added and the solution was concentrated. This process was
repeated twice and the residue was dissolved in saturated NH4Cl
(50 mL), then extracted with EtOAc (3 × 75 mL). The organic
layer was dried (MgSO4), filtered, and concentrated and the oily
residue was recrystallized from Et2O-hexanes to give the product
13 as colorless plates. The mother liquors were purified by flash
chromatography (hexanes-Et2O, 1:1 then hexanes-EtOAc, 1:1),
which gave the product 13 (4.49 g, 17.2 mmol, 75%) as a colorless
solid. Rf 0.77 (hexanes-EtOAc, 1:1); mp 73 °C{lit.13 mp 74-
75 °C}; [R]25D + 39.8 (c 0.42, CHCl3) {lit.13 [R]25D + 37.8 (c 1.0,
The oily residue was stirred in MeOH (27 mL) containing K2-
CO3 (744 mg, 5.38 mmol) for 20 min. A saturated solution of NH4-
Cl (50 mL) was added and the majority of the MeOH evaporated
before the product was extracted with CH2Cl2 (3 × 50 mL), dried
(Na2SO4), filtered, and concentrated. Flash chromatography (100%
CH2Cl2 to CH2Cl2-Et2O, 24:1) provided the product 16 (848 mg,
1.80 mmol, 58%) as a colorless gum. Rf 0.38 (hexanes-EtOAc,
1:1); [R]25 + 27.4 (c 0.95, CHCl3) {lit.26 [R]25 + 35.6 (c 0.5,
1
CHCl3)}; H NMR (400 MHz, CDCl3) δ (ppm) 5.83 (d, J ) 3.8
Hz, 1H), 4.64 (m, 1H), 4.33 (dd, J ) 11.2, 6.4 Hz, 1H), 4.07 (m,
3H), 3.85 (dd, J ) 8.5, 4.7 Hz, 1H), 2.57 (d, J ) 8.4 Hz, 1H), 1.61
(s, 3H), 1.49 (s, 3H), 1.41 (s, 3H), 1.40 (s, 3H); 13C NMR (75
MHz, CDCl3) δ (ppm) 112.4, 109.5, 103.5, 79.2, 78.6, 75.1, 72.0,
65.4, 26.2, 26.1, 25.9, 24.9; LRMS (ESI) 261 (23%) [M + H]+.
1,2-O-Isopropylidene-3-O-methyl-D-allofuranose (14). A solu-
tion of 13 (2.61 g, 10.0 mmol) in dry THF (60 mL), under Ar
atmosphere, was cooled to 0 °C and NaH (60% dispersion in
mineral oil, 600 mg, 15.0 mmol) was added portionwise. The
mixture was allowed to warm to room temperature before MeI (900
µL, 2.05 g, 14.4 mmol) was added slowly. The reaction was stirred
overnight at this temperature then cooled to 0 °C and a saturated
solution of NH4Cl (40 mL) was added. The product was extracted
with Et2O (3 × 50 mL) and the combined organic layers were
washed with brine (40 mL), dried (Na2SO4), filtered, and concen-
trated.
D
D
CHCl3)}; 1H NMR (400 MHz, CDCl3) δ (ppm) 7.70 (m, 4H), 7.40
(m, 6H), 5.79 (d, J ) 3.6 Hz, 1H), 4.70 (dd, J ) 3.8, 3.8 Hz, 1H),
4.07 (dd, J ) 9.0, 1.7 Hz, 1H), 3.90 (br s, 1H), 3.83 (m, 2H), 3.70
(dd, J ) 10.1, 5.4 Hz, 1H), 3.52 (s, 3H), 2.33 (br s, 1H), 1.57 (s,
3H), 1.38 (s, 3H), 1.07 (s, 9H); 13C NMR (75 MHz, CDCl3) δ
(ppm) 135.2, 132.7, 129.4, 127.4, 112.7, 103.9, 79.5, 77.0, 76.6,
69.3, 65.0, 58.1, 26.5, 26.2, 18.8; HRMS (ESI) calcd for C26H36O6-
SiNa [M + Na] 495.2173, found 495.2174.
5-Azido-6-O-tert-butyldiphenylsilyl-1,2-O-isopropylidene-3-O-
methyl-D-allofuranose (17). A solution of 16 (804 mg, 1.70 mmol)
in dry THF (18 mL), under Ar atmosphere, was cooled to 0 °C
and PPh3 (891 mg, 3.40 mmol) then DIAD (710 µL, 730 mg, 3.60
mmol) were added. Diphenyl phosphoryl azide (730 µL, 936 mg,
3.40 mmol) was added dropwise, and after stirring for 30 min the
mixture was warmed to room temperature. The mixture was stirred
overnight, then the solvents were evaporated under vacuum and
the product was purified by flash chromatography (hexanes-EtOAc,
9:1). Chromatography was repeated (CH2Cl2-hexanes, 7:3) to give
the product 17 (719 mg, 1.45 mmol, 85%) as a colorless oil. Rf
The oily residue was stirred in AcOH/H2O (4:1 v/v, 30 mL) for
2 days, then a saturated solution of NaHCO3 (40 mL) was added
and the mixture was neutralized by careful addition of solid
NaHCO3. After lypophilization brine (50 mL) was added and the
solution was extracted with EtOAc (5 × 60 mL), then dried
(MgSO4), filtered, and concentrated. The crude material was purified
by flash chromatography (EtOAc-hexanes, 9:1 then 100% EtOAc),
which gave the product 14 (1.76 g, 7.52 mmol, 75%) as a colorless
solid. Rf 0.18 (100% EtOAc); mp 116 °C{lit.25 mp 109-110 °C};
[R]25D +99.7 (c 1.1, CHCl3) {lit.25 [R]25D + 105.1 (c 1.0, CHCl3)};
1H NMR (400 MHz, CDCl3) δ (ppm) 5.81 (d, J ) 3.6 Hz, 1H),
4.74 (dd, J ) 3.9, 3.9 Hz, 1H), 4.08 (m, 2H), 3.83 (ddd, J ) 10.2,
4.2, 1.5 Hz, 1H), 3.73 (m, 2H), 3.53 (s, 3H), 2.51 (br s, 2H), 1.61
(s, 3H), 1.39 (s, 3H); 13C NMR (75 MHz, CDCl3) δ (ppm) 112.8,
103.7, 78.7, 78.4, 76.5, 70.4, 62.6, 57.5, 26.3, 26.1; HRMS (ESI)
calcd for C10H18O6Na [M + Na] 257.0996, found 257.0992.
6-O-tert-Butyldiphenylsilyl-1,2-O-isopropylidene-3-O-methyl-
D-allofuranose (15). To a solution of 14 (823 mg, 3.52 mmol) in
dry CH2Cl2 (18 mL), under Ar atmosphere, was added Et3N (630
µL, 457 mg, 4.52 mmol), TBDPSCl (1.00 mL, 1.07 g, 3.89 mmol),
and DMAP (cat) and the mixture was stirred at room temperature
for 30 h. The mixture was washed with a saturated solution of NH4-
Cl (2 × 15 mL), dried (Na2SO4), and filtered and the solvents were
evaporated under vacuum. The product was purified by flash
chromatography (hexanes-EtOAc, 4:1), which gave 15 (1.44 g,
0.84 (hexanes-EtOAc, 1:1); [R]25 + 33.3 (c 0.45, CHCl3); IR
D
(neat) V 2105; 1H NMR (400 MHz, CDCl3) δ (ppm) 7.70 (td, J )
7.9, 1.6 Hz, 4H), 7.42 (m, 6H), 5.78 (d, J ) 3.5 Hz, 1H), 4.66 (dd,
J ) 3.9, 3.9 Hz, 1H), 4.07 (dd, J ) 8.7, 3.5 Hz, 1H), 3.90 (m, 1H),
3.77 (m, 2H), 3.66 (dd, J ) 8.8, 4.4 Hz, 1H), 3.38 (s, 3H), 1.58 (s,
3H), 1.38 (s, 3H), 1.11 (s, 9H); 13C NMR (75 MHz, CDCl3) δ
(ppm) 135.28, 135.23, 132.6, 132.5, 129.44, 129.40, 127.40, 127.36,
112.8, 103.6, 80.1, 76.5, 71.5, 64.1, 63.3, 57.6, 26.4, 26.3, 26.1,
18.8; LRMS (ESI) 498 (8%) [M + H]+.
5-Azido-1,2-O-isopropylidene-3-O-methyl-D-allofuranose (18).
To a solution of 17 (675 mg, 1.36 mmol) in dry THF (17 mL) was
added slowly TBAF (1 M solution in THF, 2.10 mmol, 2.10 mL),
and after the mixture was stirred for 1.5 h a saturated solution of
NH4Cl (20 mL) was added. The mixture was stirred for 15 min,
the layers were separated, and the aqueous layer was extracted with
CH2Cl2 (2 × 20 mL). The organic layer was dried (Na2SO4),
filtered, and concentrated. The product was then purified by flash
chromatography (hexanes-Et2O, 65:35 then hexanes-EtOAc, 1:1),
which gave the product 18 (293 mg, 1.13 mmol, 81%) as a colorless
solid. Rf 0.52 (hexanes-EtOAc, 1:1); mp 64-65 °C; [R]25D + 295.7
1
(c 0.35, CHCl3); IR (neat) V 3478, 2102; H NMR (400 MHz,
3.05 mmol, 87%) as a colorless oil. Rf 0.54 (hexanes-EtOAc, 1:1);
CDCl3) δ (ppm) 5.85 (d, J ) 3.6 Hz, 1H), 4.76 (dd, J ) 3.6, 3.6
Hz, 1H), 4.22 (dd, J ) 8.8, 2.9 Hz, 1H), 4.02 (td, J ) 5.4, 2.9 Hz,
1H), 3.88 (dd, J ) 8.8, 4.3 Hz, 1H), 3.73 (d, J ) 5.4 Hz, 2H), 3.53
(s, 3H), 1.61 (s, 3H), 1.40 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
(ppm) 113.0, 103.6, 79.6, 77.4, 71.2, 61.6, 58.7, 57.6, 26.5, 26.1;
HRMS (ESI) calcd for C10H17N3O5Na [M + Na] 282.1060, found
282.1061.
1
[R]25 + 42.4 (c 0.85, CHCl3); H NMR (400 MHz, CDCl3) δ
D
(ppm) 7.70 (m, 4H), 7.40 (m, 6H), 5.77 (d, J ) 3.6 Hz, 1H), 4.69
(dd, J ) 3.8, 3.8 Hz, 1H), 4.02 (m, 2H), 3.79 (dd, J ) 8.3, 4.4 Hz,
1H), 3.75 (m, 2H), 3.42 (s, 3H), 2.59 (d, J ) 2.7 Hz, 1H), 1.58 (s,
3H), 1.38 (s, 3H), 1.09 (s, 9H); 13C NMR (75 MHz, CDCl3) δ
(ppm) 135.24, 135.21, 132.80, 132.76, 129.40, 129.37, 127.37,
6360 J. Org. Chem., Vol. 72, No. 17, 2007

Requirements for Fungicidal ActiVities
5-Azido-1,2-O-isopropylidene-3,6-di-O-methyl-D-allofura-
nose (19). A solution of 18 (290 mg, 1.12 mmol) in dry THF (7
mL) was cooled to 0 °C then NaH (60% dispersion in mineral oil,
72 mg, 1.79 mmol) was added portionwise. After the mixture was
stirred for 30 min, MeI (140 µL, 319 mg, 2.24 mmol) was added
dropwise, then the mixture was allowed to warm to room temper-
ature and stirred overnight. A saturated solution of NH4Cl (10 mL)
was added and the product was extracted with Et2O (3 × 10 mL),
which was dried (Na2SO4), filtered, and concentrated. The oily
residue was purified by flash chromatography (hexanes-EtOAc,
85:15), which gave the product 19 (272 mg, 1.00 mmol, 91%) as
2H), 3.70 (dd, J ) 10.3, 3.4 Hz, 1H), 3.58 (dd, J ) 10.0, 13.3 Hz,
1H), 3.43 (s, 3H), 3.39 (s, 3H), 2.20 (s, 3H), 2.14 (s, 3H); 13C
NMR (75 MHz, CD3OD) δ (ppm) 171.3, 169.6, 162.9, 160.3, 155.9,
145.1, 96.5, 90.3, 80.4, 77.7, 73.4, 71.2, 61.2, 57.5, 22.8, 18.8;
HRMS (ESI) calcd for C16H23N6O7 [M + H] 411.1623, found
411.1630.
(1′′R,2R,3R,4R,5R)-(1′′-[5-(4′-Amino-2′-oxo-2H-pyrimidin-1′-
yl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl]-2′′-methoxyethy-
l)urea (10). The N-glycoside 22 (19 mg, 0.046 mmol) was dissolved
in THF (2 mL) and PMe3 (1 M solution in THF, 170 µL, 0.17
mmol) was added. After the solution was stirred at room temper-
ature for 5 h, H2O (11 µL, 11 mg, 0.61 mmol) was added and the
mixture was heated to reflux for 74 h. The solvents were removed
and the residue was filtered through a plug of silica, washing with
EtOAc then EtOAc-MeOH (7:3). The second fraction was
collected, concentrated, and dried under vacuum overnight. The
residue was dissolved in dry CH2Cl2 (1 mL) and trichloroacetyl
isocyanate (23 µL, 36 mg, 0.18 mmol) was added under an Ar
atmosphere at room temperature. LCMS showed the consumption
of the intermediate amine after 1.5 h, so the solvents were
evaporated and the residue was stirred in MeNH2 40 wt % in H2O/
MeOH (3:1, 0.6 mL) for 1 h. The solution was then lypophilized
and the residue was purified by preparative thin layer chromatog-
raphy (CHCl3-MeOH, 4:1 to 7:3) to provide the product 10 (7
mg, 0.020 mmol, 44%) as a colorless solid. Rf 0.11 (CHCl3-MeOH,
7:3); [R]25D -32.0 (c 0.35, MeOH); 1H NMR (400 MHz, CD3OD)
δ (ppm) 7.66 (d, J ) 7.5 Hz, 1H), 5.96 (d, J ) 7.5 Hz, 1H), 5.93
(d, J ) 6.0 Hz, 1H), 4.30 (dd, J ) 5.8, 5.8 Hz, 1H), 4.03 (m, 2H),
3.84 (dd, J ) 5.5, 3.4 Hz, 1H), 3.59 (dd, J ) 9.4, 3.5 Hz, 1H),
3.47 (dd, J ) 9.6, 3.5 Hz, 1H), 3.44 (s, 3H), 3.38 (s, 3H); 13C
NMR (75 MHz, CD3OD) δ (ppm) 165.8, 159.9, 156.8, 140.8, 94.8,
89.3, 80.8, 79.8, 72.3, 71.1, 57.6, 56.5, 50.6; HRMS (ESI) calcd
for C13H22N5O6 [M + H] 344.1565, found 344.1562.
a colorless oil. Rf 0.70 (hexanes-EtOAc, 1:1); [R]25 + 107.6 (c
D
1
2.25, CHCl3); IR (neat) V 2099; H NMR (400 MHz, CDCl3) δ
(ppm) 5.81 (d, J ) 3.6 Hz, 1H), 4.72 (dd, J ) 4.1, 3.7 Hz, 1H),
4.10 (m, 1H), 4.02 (dt, J ) 9.7, 3.5 Hz, 1H), 3.80 (dd, J ) 8.7, 4.3
Hz, 1H), 3.52 (dd, J ) 10.3, 3.9 Hz, 1H), 3.49 (s, 3H), 3.42 (dd,
J ) 10.3, 8.7 Hz, 1H), 3.41 (s, 3H), 1.61 (s, 3H), 1.39 (s, 3H); 13
C
NMR (75 MHz, CDCl3) δ (ppm) 113.0, 103.6, 79.6, 77.5, 76.7,
71.2, 61.6, 58.7, 57.6, 26.5, 26.1; HRMS (ESI) calcd for C11H19N3O5-
Na [M + Na] 296.1217, found 296.1213.
5-Azido-1,2-di-O-acetyl-3,6-di-O-methyl-D-allofuranose (21).
Compound 19 (84 mg, 0.31 mmol) was stirred in TFA/H2O (1:1,
v/v, 1 mL) for 2 h, a saturated solution of NaHCO3 (3 mL) was
added, and the mixture was neutralized with solid NaHCO3. The
product was extracted with EtOAc (3 × 5 mL), dried (Na2SO4),
filtered, and concentrated. The crude material was used in the next
step.
The oily residue was dissolved in anhydrous pyridine/anhydrous
CH2Cl2 (2:1, 1.2 mL) and Ac2O (290 µL, 313 mg, 3.07 mmol)
then DMAP (cat.) were added. The mixture was stirred at room
temperature overnight. After this time the solvents were evaporated
and the crude material was purified by flash chromatography
(hexanes-EtOAc, 4:1 to 7:3) to provide the product 21 (80 mg,
0.25 mmol, 81%) as a colorless oil and a 1:1 mixture of anomers.
3-O-Methyl-1,2:5,6-di-O-isopropylidene-3-C-prop-1′-enyl-D-
allofuranose (29). The alcohol 2321 (3.67 g, 12.2 mmol) was
dissolved in dry THF (75 mL), under an Ar atmosphere, and NaH
(60% dispersion in mineral oil, 732 mg, 18.3 mmol) was added
portionwise. The mixture was then refluxed gently for 1.5 h before
being cooled to room temperature when HMPA (3 mL) and MeI
(1.55 mL, 3.53 g, 24.9 mmol) were added. The mixture was heated
to reflux for a further 2 h, then cooled to room temperature, and a
saturated solution of NH4Cl/ice (50 mL/20 g) was added. The
product was extracted with Et2O (3 × 50 mL), dried (Na2SO4),
filtered, and concentrated to give an oily residue. Purification by
flash chromatography (hexanes-Et2O, 4:1) provided the product
29 (3.59 g, 11.4 mmol, 94%) as a very pale yellow oil. Rf 0.19
(hexanes-Et2O, 4:1); [R]25 + 50.4 (c 0.54, CHCl3) {lit.21 [R]25
â-Anomer: Rf 0.68 (hexanes-EtOAc, 1:1); [R]25 + 47.4 (c 2.0,
D
CHCl3); IR (neat) V 2133, 2100, 1749; 1H NMR (400 MHz, CDCl3)
δ (ppm) 6.12 (s, 1H), 5.31 (d, J ) 3.9 Hz, 1H), 4.12 (m, 2H), 3.91
(m, 1H), 3.48 (dd, J ) 10.3, 4.3 Hz, 1H), 3.38 (s, 3H), 3.37 (s,
3H), 3.31 (dd, J ) 10.4, 7.9 Hz, 1H), 2.15 (s, 3H), 2.10 (s, 3H);
13C NMR (75 MHz, CDCl3) δ (ppm) 169.3, 168.7, 97.7, 80.4, 78.0,
72.8, 70.9, 62.0, 58.8, 58.5, 20.7, 20.3; LRMS (ESI) 258 (100%)
[M - OAc]+.
R-Anomer: Rf 0.58 (hexanes-EtOAc, 1:1); [R]25 + 58.5 (c
D
1
1.85, CHCl3); IR (neat) V 2134, 2101, 1749; H NMR (400 MHz,
CDCl3) δ (ppm) 6.39 (d, J ) 4.6 Hz, 1H), 5.08 (dd, J ) 6.7, 4.7
Hz, 1H), 4.24 (dd, J ) 4.6, 3.4 Hz, 1H), 3.96 (dd, J ) 6.6, 3.4 Hz,
1H), 3.75 (dt, J ) 7.0, 4.8 Hz, 1H), 3.59 (dd, J ) 10.2, 4.7 Hz,
1H), 3.50 (dd, J ) 10.2, 7.0 Hz, 1H), 3.40 (s, 3H), 3.39 (s, 3H),
2.16 (s, 3H), 2.13 (s, 3H); 13C NMR (75 MHz, CDCl3) δ (ppm)
169.7, 169.6, 93.5, 83.0, 76.8, 71.5, 70.9, 61.6, 58.9, 58.6, 20.8,
20.2; LRMS (ESI) 258 (100%) [M - OAc]+.
D
D
1
+20.0 (c 1.56, CHCl3)}; H NMR (400 MHz, CDCl3) δ (ppm)
5.97 (m, 1H), 5.59 (d, J ) 3.5 Hz, 1H), 5.20 (m, 2H), 4.43 (d, J )
3.6 Hz, 1H), 4.12 (m, 3H), 3.91 (m, 1H), 3.51 (s, 3H), 2.71 (dd, J
) 15.0, 6.2 Hz, 1H), 2.30 (dd, J ) 15.1, 7.7 Hz, 1H), 1.61 (s, 3H),
1.46 (s, 3H), 1.39 (s, 3H), 1.36 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ (ppm) 132.0, 118.3, 112.3, 109.4, 102.6, 83.2, 82.8, 81.7, 72.3,
68.2, 52.6, 33.7, 26.7, 26.13, 26.06, 25.1; LRMS (ESI) 315 (6%)
[M + H]+.
(2R,3R,4R,5R,1′’R)-Acetic Acid 2-(4′-Acetylamino-2′-oxo-2H-
pyrimidin-1′-yl)-5-(1′′-azido-2′′-methoxyethyl)-4-methoxytet-
rahydrofuran-3-yl Ester (22). To a solution of crude bis-silylated
N-acetyl cytosine17b (1.50 mmol of crude material) in anhydrous
dichloroethane (2 mL), under Ar atmosphere, was added a solution
of 21 (70 mg, 0.22 mmol) in anhydrous dichloroethane at room
temperature. SnCl4 (51 µL, 114 mg, 0.44 mmol) was added
dropwise to the solution. Additional SnCl4 (100 µL, 223 mg, 0.86
mmol) was added in 2 portions at 12 h intervals and the solution
was stirred a further 12 h. A saturated solution of NaHCO3 (5 mL)
was added, and the product was extracted with EtOAc (3 × 10
mL), dried (MgSO4), filtered, and concentrated. The crude material
was purified by preparative thin layer chromatography (100%
EtOAc) to provide the product 22 (61 mg, 0.15 mmol, 67%) as a
3-O-Methyl-1,2:5,6-di-O-isopropylidene-3-C-(1′-hydroxypro-
pyl)-D-allofuranose (30). Compound 29 (3.52 g, 11.2 mmol) was
dissolved in CH2Cl2 (90 mL) and the solution was cooled to
-78 °C. Ozone was bubbled through the solution until an excess
was present and the mixture was allowed to stir for 10 min. The
ozone was removed by sparging the solution with O2, then NaBH4
(3.90 g, 103 mmol) was added followed by careful addition of
MeOH (40 mL). The solution was allowed to warm to room
temperature, and was then stirred for 24 h. A saturated solution of
NH4Cl (50 mL) was added, and stirring was continued for 10 min,
after which time the majority of the MeOH was removed under
vacuum. Water (20 mL) was added and the aqueous layer was
extracted with EtOAc (3 × 70 mL), dried (MgSO4), filtered, and
concentrated. The residue was purified by flash chromatography
colorless foam. Rf 0.44 (100% EtOAc); [R]25 + 92.7 (c 0.75,
D
1
MeOH); H NMR (400 MHz, CD3OD) δ (ppm) 8.17 (d, J ) 7.6
Hz, 1H), 7.47 (d, J ) 7.5 Hz, 1H), 5.93 (d, J ) 3.4 Hz, 1H), 5.52
(dd, J ) 5.5, 3.4 Hz, 1H), 4.18 (dd, J ) 5.7, 5.7 Hz, 1H), 4.09 (m,
J. Org. Chem, Vol. 72, No. 17, 2007 6361

Loiseleur et al.
(hexanes-EtOAc, 1:1) to provide the product 30 (2.73 g, 8.58
repeated and the residue was dissolved in water (5 mL), then the
product was extracted with CH2Cl2 (3 × 5 mL), dried (Na2SO4),
filtered, and concentrated.
mmol, 77%) as a colorless, viscous oil. Rf 0.16 (hexanes-EtOAc,
1
1:1); [R]25 + 37.1 (c 0.75, CHCl3); IR (neat) V 3500; H NMR
D
(400 MHz, CDCl3) δ (ppm) 5.62 (d, J ) 3.6 Hz, 1H), 4.54 (d, J )
3.5 Hz, 1H), 4.13 (m, 3H), 3.90 (m, 3H), 3.54 (s, 3H), 2.26 (br s,
1H), 2.09 (dt, J ) 14.9, 6.5 Hz, 1H), 1.75 (dt, J ) 15.0, 6.1 Hz,
1H), 1.60 (s, 3H), 1.44 (s, 3H), 1.37 (s, 3H), 1.36 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ (ppm) 112.6, 109.5, 102.5, 83.8, 83.5,
80.3, 72.5, 68.5, 58.0, 53.1, 31.8, 26.7, 26.1, 26.0, 25.0; HRMS
(ESI) calcd for C15H26O7Na [M + Na] 341.1571, found 341.1572.
3-O-Methyl-1,2:5,6-di-O-isopropylidene-3-C-(1′-azidopropyl)-
D-allofuranose (31). Methanesulfonyl chloride (58 µL, 86 mg, 0.75
mmol) was added to a solution of 30 (200 mg, 0.63 mmol) and
Et3N (140 µL, 102 mg, 1.00 mmol) in anhydrous CH2Cl2 (5 mL)
at 0 °C. After 1 h, water (5 mL) was added and the mixture was
stirred for 10 min. The layers were separated and the organic phase
was washed with a saturated solution of NH4Cl (5 mL), dried (Na2-
SO4), filtered, and concentrated.
To a solution of the crude oil in anhydrous CH2Cl2 (4 mL), under
Ar atmosphere, at 0 °C was added Et3N (81 µL, 59 mg, 0.58 mmol)
and methanesulfonyl chloride (39 µL, 57 mg, 0.50 mmol). After 1
h, water (5 mL) was added and the solution was stirred for 20 min.
The product was extracted with CH2Cl2 (3 × 5 mL), dried (Na2-
SO4), filtered, and concentrated. The crude material was purified
by flash chromatography (hexanes-EtOAc, 7:3) to provide the
product 36 (135 mg, 0.36 mmol, 86%) as a colorless oil. Rf 0.54
(hexanes-EtOAc, 1:1); [R]25D +45.6 (c 0.95, CHCl3); IR (neat) V
1
2100; H NMR (400 MHz, CDCl3) δ (ppm) 5.74 (d, J ) 3.6 Hz,
1H), 4.43 (d, J ) 3.7 Hz, 1H), 4.41 (dd, J ) 10.5, 1.6 Hz, 1H),
4.33 (m, 1H), 4.28 (dd, J ) 10.4, 7.7 Hz, 1H), 3.44 (m, 5H), 3.09
(s, 3H), 1.94 (ddd, J ) 14.9, 8.9, 6.3 Hz, 1H), 1.75 (ddd, J ) 14.9,
8.9, 6.2 Hz, 1H), 1.59 (s, 3H), 1.36 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ (ppm) 112.9, 103.1, 82.5, 81.8, 77.9, 67.3, 52.7, 45.5,
37.5, 28.5, 26.4, 26.0; HRMS (ESI) calcd for C12H21N3O7SNa [M
+ Na] 351.0992, found 351.0995.
(2R,3R,3aR,7aR)-2,3-O-Isopropylidene-3a-methoxy-6-(2′-me-
thylpropane-2′-sulfonyl)octahydrofuro[2,3-c]pyridine (37). A
suspension of 36 (135 mg, 0.38 mmol) and Pd black (18 mg) in
MeOH/Et3N (99:1, 5 mL) was degassed and an H2 atmosphere (1
atms) was applied. After the solution was stirred for 1 h at room
temperature, TLC indicated the consumption of starting material,
so the suspension was filtered and the residue was washed with
hot MeOH. The solvents were evaporated under vacuum and the
residue was used directly in the next step.
The crude amine was dissolved in MeCN (5 mL) and Et3N (210
µL, 152 mg, 1.50 mmol) then tert-butylsulfinyl chloride (1 M in
CH2Cl2,490 µL, 0.49 mmol) was added at room temperature. After
2.5 h, water (5 mL) was added and stirring was continued for 10
min. The majority of the MeCN was removed under vacuum and
brine (4 mL) was added. The product was extracted with EtOAc
(3 × 8 mL), dried (MgSO4), filtered, and concentrated. The excess
triethylamine was removed by filtration through silica, washing with
EtOAc-hexanes (4:1) after which the solvents were evaporated.
The orange syrup was dissolved in CH2Cl2 (4 mL) and m-CPBA
(91 mg, 0.53 mmol) was added to the solution at room temperature.
After the mixtue was stirred overnight, a saturated solution of Na2-
SO3 (4 mL) was added and stirring was continued for a further 30
min before the product was extracted with CH2Cl2 (2 × 5 mL).
The combined organic layers were washed with a saturated solution
of NaHCO3 (2 × 5 mL), dried (Na2SO4), filtered, and concentrated.
The crude material was purified by flash chromatography (hexanes-
EtOAc, 65:35) to provide the product 37 (63 mg, 0.18 mmol, 48%)
The residue was dissolved in dry DMF (4 mL) and NaN3 (59
mg, 0.91 mmol) was added before the solution was heated to 90
°C. After 1 h the solution was cooled to room temperature and
poured into brine (5 mL). The product was extracted with Et2O (3
× 8 mL), which was then washed with H2O (2 × 5 mL), dried
(Na2SO4), and filtered, and the solvent was evaporated under
vacuum. The crude material was purified by flash chromatography
(hexanes-EtOAc, 9:1) to provide the product 31 (186 mg, 0.54
mmol, 86%) as a colorless oil. Rf 0.77 (hexanes-EtOAc, 1:1);
1
[R]25 -16.2 (c 1.18, CHCl3); IR (neat) V 2099; H NMR (400
D
MHz, CDCl3) δ (ppm) 5.63 (d, J ) 3.4 Hz, 1H), 4.45 (d, J ) 3.5
Hz, 1H), 4.16 (dd, J ) 8.3, 5.9 Hz, 1H), 4.06 (d, J ) 9.0 Hz, 1H),
4.00 (ddd, J ) 9.0, 6.2, 5.9 Hz, 1H), 3.88 (dd, J ) 8.4, 6.2 Hz,
1H), 3.57 (m, 2H), 3.50 (s, 3H), 2.09 (ddd, J ) 14.7, 8.8, 5.9 Hz,
1H), 1.80 (m, 1H), 1.60 (s, 3H), 1.45 (s, 3H), 1.38 (s, 3H), 1.36 (s,
3H); 13C NMR (75 MHz, CDCl3) δ (ppm) 112.7, 109.6, 102.4,
83.2, 83.1, 80.9, 72.4, 68.5, 52.8, 45.8, 28.6, 26.6, 26.07, 26.05
25.1; HRMS (ESI) calcd for C15H25N3O6Na [M + Na] 366.1635,
found 366.1631.
3-O-Methyl-1,2-O-isopropylidene-3-C-(1′-azidopropyl)-D-al-
lofuranose (32). The acetonide 31 (186 mg, 0.54 mmol) was stirred
in AcOH/H2O (4:1, v/v, 2 mL) for 3 days, then a saturated solution
of NaHCO3 (3 mL) was added and the mixture was neutralized by
careful addition of solid NaHCO3. The product was extracted with
Et2O (3 × 6 mL), dried (Na2SO4), filtered, and concentrated. The
crude product was purified by flash chromatography (hexanes-
EtOAc, 1:1) to provide the product 32 (135 mg, 0.45 mmol, 83%)
as a colorless, viscous oil. Rf 0.18 (hexanes-EtOAc, 1:1); [R]25
D
1
+ 61.7 (c 0.56, CHCl3); IR (neat) V 3467, 2100; H NMR (400
MHz, CDCl3) δ (ppm) 5.67 (d, J ) 3.6 Hz, 1H), 4.43 (d, J ) 3.6
Hz, 1H), 4.02 (d, J ) 8.8 Hz, 1H), 3.82 (dd, J ) 11.0, 3.5 Hz,
1H), 3.76 (ddd, J ) 8.8, 4.5, 3.5 Hz, 1H), 3.67 (dd, J ) 10.9, 4.5
Hz, 1H), 3.57 (m, 2H), 3.49 (s, 3H), 2.39 (br s, 2H), 2.07 (ddd, J
) 14.9, 8.8, 6.9 Hz, 1H), 1.92 (ddd, J ) 15.1, 9.2, 6.2 Hz, 1H),
1.60 (s, 3H), 1.37 (s, 3H); 13C NMR (75 MHz, CDCl3) δ (ppm)
112.9, 103.0, 83.8, 82.2, 78.0, 69.4, 64.3, 52.9, 46.0, 29.0, 26.4,
26.1; HRMS (ESI) calcd for C12H21N3O6Na [M + Na] 326.1323,
found 326.1322.
(2R,3R,4R,5R)-Methanesulfonic Acid 3-(2′-Azidoethyl)-4,5-
O-isopropylidene-3-methoxytetrahydrofuran-2-ylmethyl Ester
(36). To a stirred solution of 32 (135 mg, 0.45 mmol) in CH2Cl2/
MeOH/H2O (1:1:1, 5 mL) at room temperature was added NaIO4
(123 mg, 0.58 mmol). After 45 min, water (5 mL) was added and
the product was extracted with CH2Cl2 (3 × 5 mL). The organic
layer was then washed with water (5 mL), dried (Na2SO4), filtered,
and concentrated, and the crude material was used directly in the
next step.
as a colorless solid. Rf 0.34 (hexanes-EtOAc, 1:1); mp 132 °C;
1
[R]25 + 30.3 (c 1.55, CHCl3); H NMR (400 MHz, CDCl3) δ
D
(ppm) 5.77 (d, J ) 3.7 Hz, 1H), 4.26 (d, J ) 3.6 Hz, 1H), 3.95 (m,
2H), 3.62 (d, J ) 2.8 Hz, 1H), 3.38 (s, 3H), 3.20 (dd, J ) 14.5, 2.0
Hz, 1H), 3.07 (td, J ) 13.1, 2.0 Hz, 1H), 1.96 (d, J ) 4.4 Hz, 1H),
1.57 (s, 3H), 1.34 (m, 13H); 13C NMR (75 MHz, CDCl3) δ (ppm)
112.6, 103.8, 80.4, 78.2, 74.1, 61.0, 51.5, 44.8, 41.2, 26.3, 26.0,
25.1, 24.0; LRMS (ESI) 350 (100%) [M + H]+.
(2R,3R,3aR,7aR)-3a-Methoxy-6-(2′-methylpropane-2′-sulfonyl)-
2-phenylsulfanyloctahydrofuro[2,3-c]pyridin-3-ol (38). To a
solution of 37 (171 mg, 0.49 mmol) and thiophenol (504 µL, 540
mg, 4.91 mmol) in anhydrous CH2Cl2 (12 mL), under an Ar
atmosphere, at room temperature was added Amberlyst-15 (350
mg). The suspension was stirred for 10 days, filtered, washed with
CH2Cl2, and concentrated. The crude material was purified by flash
chromatography (hexanes-EtOAc, 3:2 to 1:1) to provide the
product 38 (97 mg, 0.24 mmol, 49%) as a colorless solid and a 5:1
mixture of R:â-anomers, respectively. The minor â-anomer was
The oily residue was dissolved in MeOH (4 mL) and NaBH4
(29 mg, 0.77 mmol) was added at room temperature. The solution
was stirred for 45 min then the solvent was evaporated under
vacuum and MeOH (10 mL) was added. This procedure was
not characterized. Rf 0.22 (hexanes-EtOAc, 1:1); mp 165 °C; [R]25
D
1
+161.7 (c 0.90, CHCl3); IR (neat) V 3459; H NMR (400 MHz,
CDCl3) δ (ppm) 7.53 (m, 2H), 7.25 (m, 3H), 5.68 (d, J ) 3.5 Hz,
1H), 4.15 (m, 1H), 4.06 (s, 1H), 3.95 (br s, 1H), 3.65 (br s, 1H),
6362 J. Org. Chem., Vol. 72, No. 17, 2007

Requirements for Fungicidal ActiVities
3.40 (s, 3H), 3.30 (dd, J ) 14.5, 2.5 Hz, 1H), 3.14 (m, 1H), 2.81
(d, J ) 4.6 Hz, 1H), 2.13 (d, J ) 15.2 Hz, 1H) 1.63 (ddd, J )
15.0, 12.0, 4.4 Hz, 1H), 1.40 (s, 9H); 13C NMR (75 MHz, CDCl3)
δ (ppm) 135.1, 130.5, 128.5, 126.7, 92.4, 78.4, 74.1, 73.4, 61.1,
51.5, 45.5, 41.7, 25.2, 24.0; HRMS (ESI) calcd for C18H27N5O5S2-
Na [M + Na] 424.1223, found 424.1219.
EtOAc, 4:1 to 1:4) to provide the product 40 (33 mg, 0.063 mmol,
76%) as a light orange solid. Rf 0.35 (100% EtOAc); mp >210 °C
1
dec; [R]25 + 18.2 (c 1.60, CHCl3); H NMR (400 MHz, CDCl3)
D
δ (ppm) 8.89 (br s, 1H), 8.40 (d, J ) 4.7 Hz, 1H), 7.55 (d, J ) 4.8
Hz, 1H), 5.74 (s, 1H), 5.11 (s, 1H), 4.22 (d, J ) 9.6 Hz, 1H), 3.97
(s, 1H), 3.67 (d, J ) 8.8 Hz, 1H), 3.39 (dd, J ) 9.5, 1.1 Hz, 1H),
3.20 (s, 3H), 3.10 (t, J ) 8.1 Hz, 1H), 2.28 (s, 3H), 2.06 (d, J )
6.4 Hz, 1H), 1.41 (s, 9H), 1.39 (m, 1H), 1.32 (s, 9H); 13C NMR
(75 MHz, CDCl3) δ (ppm) 175.9, 170.8, 162.9, 154.3, 143.6, 96.6,
91.5, 76.9, 76.5, 75.8, 61.1, 52.3, 44.8, 42.0, 38.5, 26.7, 26.3, 24.6,
24.1; LRMS (ESI) 529 (100%) [M + H]+.
(2R,3R,3aR,7aR)-2′′,2′′-Dimethylpropionic Acid 3a-Methoxy-
6-(2′-methylpropane-2′-sulfonyl)-2-phenylsulfanyloctahydrofuro-
[2,3-c]pyridin-3-yl Ester (39). The alcohol 38 (80 mg, 0.20 mmol)
was dissolved in anhydrous pyridine/anhydrous CH2Cl2 (2:1, 3 mL),
under an Ar atmosphere, and pivaloyl chloride (250 µL, 245 mg,
2.03 mmol) then DMAP (24 mg, 0.20 mol) were added at room
temperature. After the mixture was stirred for 8 h, the solvents were
evaporated under vacuum and the residue was dissolved in CH2-
Cl2 (8 mL), which was washed with 0.1 M HCl (5 mL) then H2O
(5 mL). The organic phase was dried (Na2SO4), filtered, and
concentrated then purified by flash chromatography (hexanes-
EtOAc, 4:1) to provide the product 39 (82 mg, 0.17 mmol, 85%)
as a colorless solid. Rf 0.89 (hexanes-EtOAc, 1:1); [R]25D +122.9
(2R,3R,3aR,7aR)-2-(4′-Amino-2′-oxo-2H-pyrimidin-1′-yl)-3-
hydroxy-3a-methoxyhexahydrofuro[2,3-c]pyridine-6-carboxy-
lic Acid Amide (11). To a solution of 40 (31 mg, 0.059 mmol)
and anisole (96 µL, 95 mg, 0.88 mmol) in anhydrous CH2Cl2 (0.75
mL) was added TfOH (13 µL, 21 mg, 0.14 mmol) dropwise. When
TLC indicated the disappearance of starting material, trichloroacetyl
isocyanate (28 µL, 44 mg, 0.24 mmol) and anhydrous pyridine (10
µL, 10 mg, 0.12 mmol) were added dropwise. When LCMS
indicated consumption of the intermediate amine, the solvents were
removed under vacuum (without heating) and the residue was
dissolved in 40 wt % MeNH2 in H2O/MeOH (2:1, 1.2 mL). After
the solution was stirred for 3 days, the solvents were removed and
the residue was purified by preparative HPLC to provide the product
40 (13 mg, 0.040 mmol, 68%) as a colorless solid. Rf 0.19 (CHCl3-
MeOH, 4:1); [R]25D + 107.5 (c 0.60, MeOH); 1H NMR (400 MHz,
CD3OD) δ (ppm) 7.86 (d, J ) 8.0 Hz, 1H), 6.14 (d, J ) 7.7 Hz,
1H), 5.70 (s, 1H), 4.19 (d, J ) 5.0 Hz, 1H), 4.14 (s, 1H), 4.08 (s,
1H), 3.74 (br s, 1H), 3.40 (br s, 4H), 3.15 (br s, 1H), 2.11 (d, J )
14.8 Hz, 1H), 1.29 (br s, 1H); 13C NMR (75 MHz, CD3OD) δ (ppm)
159.6, 159.5, 147.1, 142.9, 93.2, 92.3, 78.0, 77.3, 75.5, 50.3, 42.9,
38.7, 24.4; HRMS (ESI) calcd for C13H20N5O5 [M + H] 326.1459,
found 326.1456.
1
(c 3.0, CHCl3); IR (neat) V 1737; H NMR (400 MHz, CDCl3) δ
(ppm) 7.52 (dd, J ) 7.9, 1.2 Hz, 2H), 7.28 (m, 3H), 5.72 (d, J )
4.6 Hz, 1H), 5.42 (d, J ) 4.5 Hz, 1H), 4.10 (s, 1H), 4.03 (d, J )
4.6 Hz, 1H), 3.67 (d, J ) 4.6 Hz, 1H), 3.26 (dd, J ) 14.6, 2.0 Hz,
1H), 3.19 (s, 3H), 3.04 (m, 1H), 2.10 (d, J ) 14.9 Hz, 1H), 1.59
(m, 1H) 1.39 (s, 9H), 1.35 (s, 9H); 13C NMR (75 MHz, CDCl3) δ
(ppm) 176.6, 134.8, 130.6, 128.6, 126.9, 90.1, 77.8, 75.3, 73.3,
61.1, 52.1, 45.0, 41.4, 39.1, 26.9, 25.1, 24.0; HRMS (ESI) calcd
for C23H35NO6S2Na [M + Na] 508.1798, found 508.1791.
(2R,3R,3aR,7aR)-2′′,2′′-Dimethylpropionic Acid 2-(4′-Acety-
lamino-2′-oxo-2H-pyrimidin-1′-yl)-3a-methoxy-6-(2′′-methylpro-
pane-2′′-sulfonyl)octahydrofuro[2,3-c]pyridin-3-yl Ester (40).
Compound 39 (40 mg, 0.082 mmol) in anhydrous CH2Cl2 (1.4 mL)
was added to a solution of the crude bis-silylated N-acetyl
cytosine17b (0.57 mmol of crude material), under Ar atmosphere,
in anhydrous CH2Cl2 (1 mL) at room temperature. NIS (74 mg,
0.33 mmol, lypophilized overnight) was added, then a solution of
TfOH (10 µL, 17 mg, 0.11 mmol) in anhydrous CH2Cl2 (0.2 mL)
was added dropwise. After 20 h, a saturated solution of Na2S2O3
(4 mL) was added and the solution was stirred for 30 min before
a saturated solution of NaHCO3 (1.5 mL) was added. The product
was extracted with EtOAc (4 × 8 mL), dried (MgSO4), filtered,
and concentrated. The residue was dissolved in CH2Cl2 (3 mL),
then stirred with a saturated solution of Na2S2O3 (3 mL) for 20
min. The layers were separated and the organics washed with 0.1
M HCl (2 × 3 mL), dried (Na2SO4), filtered, and concentrated.
The crude material was purified by flash chromatography (hexanes-
Acknowledgment. We thank NSERC and Syngenta for
generous financial support. We also thank Dr. Michel Simard
for X-ray crystal structure determination.
Supporting Information Available: 1H NMR and 13C NMR
spectra of compounds 13-19, 21, 22, 10, 29-32, 36-40, and 11
and X-ray crystallographic data for compounds 18 and 44. This
material is available free of charge via the Internet at http://pubs.
acs.org.
JO070520D
J. Org. Chem, Vol. 72, No. 17, 2007 6363