A scalable synthesis of 1-cytosinyl-N-malayamycin A: A potent fungicide

Article abstract of DOI:10.1021/op0600299

A stereocontrolled synthesis of 1-cytosinyl-N-malayamycin A, an N-analogue of the naturally occurring malayamycin A with fungicidal activity, is reported. The approach was designed to rely solely on substrate control for introduction of the required stere

Full text of DOI:10.1021/op0600299

Organic Process Research & Development 2006, 10, 518−524
A Scalable Synthesis of 1-Cytosinyl-N-malayamycin A: A Potent Fungicide
Olivier Loiseleur,*,† Hermann Schneider,† Guobin Huang,‡ Roger Machaalani,‡ Patrice Selle`s,† Patrick Crowley,† and
Stephen Hanessian*,‡
Research Chemistry, Syngenta Crop Protection AG, CH-4002 Basel, Switzerland
Abstract:
A stereocontrolled synthesis of 1-cytosinyl-N-malayamycin A,
an N-analogue of the naturally occurring malayamycin A with
fungicidal activity, is reported. The approach was designed to
rely solely on substrate control for introduction of the required
stereochemistry, avoiding the use of chiral reagents or auxil-
iaries. Formation of the N-nucleoside was achieved through the
activation of a thioglycoside, proceeding via sulfonium and
thionium intermediates. Ring closure metathesis was used to
build the bicyclic perhydrofuropyran heterocycle.
Introduction
Malayamycin A (1, Figure 1) is a naturally occurring
C-nucleoside discovered at Syngenta Crop Protection Labo-
ratories in Jealott’s Hill, U.K. It was isolated from the soil
organism Streptomyces malaysiensis, and its structure was
determined by extensive NMR studies and degradative work.1
Only a handful of N- and C-nucleosides (1 and 5-7, Figure
1) feature a bicyclic perhydrofuran motif2 rather than the
commonly encountered monocyclic pentofuranosyl and
hexofuranosyl core.3 Malayamycin A is the most recent entry
in this group. In line with the ezomycins that exhibit
* Corresponding authors: Syngenta AG, Crop Protection Research
&
Technology, WRO-1060.5.04, CH-4002 Basel. E-mail: ol215@cam.ac.uk;
Department of Chemistry, Universite´ de Montre´al, C. P. 6128, Succ.
Centre-Ville,
stephen.hanessian@umontreal.ca.
Montre´al,
P.
Q.,
Canada,
H3C
3J7.
E-mail:
Figure 1. Structures of N- and C-nucleosides with a bicyclic
dioxa perhydrofuropyran motif.
† Current address: Syngenta AG, Crop Protection Research & Technology,
Schwarzwaldallee 215, CH-4002 Basel, Switzerland.
‡ Current address: Department of Chemistry, Universite´ de Montre´al, C. P.
6128, Succ. Centre-Ville, Montre´al, P. Q., Canada, H3C 3J7.
antifungal and antibiotic activities,2b,f malayamycin A is a
potent fungicide even though it displays a lower molecular
weight. Recently, the proposed structure and absolute ster-
eochemistry of 1 were confirmed by a stereocontrolled total
synthesis.4,5 By analogy with the naturally occurring N- and
C-ezomycins, it was of interest to consider N-malayamycin
A (2) and the related pyrimidine and purine congeners (3, 4
Figure 1) as potential targets. The availability of such
analogues with modified heterocyclic aglycones would
provide an opportunity to test their biological activities, since
the corresponding C-malayamycins are presently not avail-
able. Accordingly, a synthetic route to N-malayamycins
allowing for anomeric diversity was developed.6,7 Biological
screening then showed the 1-cytosinyl-N-nucleoside 3 to be
(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
app. No. WO2003062242, July 31, 2003.
(2) For isolation and structure of ezomycins, see: (a) Sakata, K.; Sakurai, A.;
Tamura, S. Agric. Biol. Chem. 1973, 37, 697. (b) Sakata, K.; Sakurai, A.;
Tamura, S. Agric. Biol. Chem. 1974, 38, 1883. (c) Sakata, K.; Sakurai, A.;
Tamura, S. Tetrahedron Lett. 1974, 4327. (d) Sakata, K.; Sakurai, A.;
Tamura, S. Tetrahedron Lett. 1975, 3191. (e) Sakata, K.; Sakurai, A.;
Tamura, S. Agric. Biol. Chem. 1976, 40, 1993. (f) Sakata, K.; Sakurai, A.;
Tamura, S. Agric. Biol. Chem. 1977, 41, 2027. (g) Sakata, K.; Sakurai, A.;
Tamura, S. Agric. Biol. Chem. 1977, 41, 2033. For isolation and structure
of octosyl acid A, see: (h) Isono, K.; Crain, P.-F.; McCloskey, J. A. J. Am.
Chem. Soc. 1975, 97, 943. For the total synthesis of octosyl acid A, see:
(i) Danishefsky, S.; Hungate, R. J. Am. Chem. Soc. 1986, 108, 2486. (j)
Hanessian, S.; Kloss, J.; Sugawara, T. J. Am. Chem. Soc. 1986, 108, 2758.
The perhydrofuropyran motif was also revealed in the structure of
miharamycin and is embedded in the “man made” quantamycin: (k) Seto,
H.; Koyama, M.; Ogino, H.; Tsuruoka, T. Tetrahedron Lett. 1983, 24, 1805.
(l) Hanessian, S.; Sato, K.; Liak, T. J.; Danh, N.; Dixit, D.; Cheney, B. V.
J. Am. Chem. Soc. 1984, 106, 6114. For synthetic studies related to the
perhydrofuropyran part of the ezomycins, see: (3c), (10b) and references
cited in (m) Khalaf, J. K.; Datta, A. J. Org. Chem. 2005, 70, 6937.
(3) For reviews, see: (a) Knapp, S. Chem. ReV. 1995, 95, 1859. (b) Isono, K.
J. Antibiot. 1988, 41, 1711. (c) Hanessian, S.; Dixit, D.-M.; Liak, T.-J. Pure
Appl. Chem. 1981, 53, 129.
(4) Hanessian, S.; Machaalani, R.; Marcotte, S. Patent app. No. WO 2004069842,
August 19, 2004.
(5) Hanessian, S.; Marcotte, S.; Machaalani, R.; Huang, G. Org. Lett. 2003, 5,
4277.
(6) Hanessian, S.; Huang, G.; Chenel, C.; Machaalani, R.; Loiseleur, O. J. Org.
Chem. 2005, 70, 6721.
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10.1021/op0600299 CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/27/2006

Scheme 1. Retrosynthetic analysis of 3
was to maintain the integrity of the trans-fused and relatively
strained bicyclic core during the exchange of a thiophenyl
group to a purine or pyrimidine moiety.
Synthesis of the Perhydrofuropyran Motif 9. Our
efforts first revolved around the stereocontrolled construction
of the trans-fused bicyclic dioxa core. The synthesis of 9
commenced with the known 1,2,5,6-di-O-ispropilydene-R-
D-allofuranose 12, which is available in two steps from
diacetone-D-glucose or can be obtained from a commercial
supplier.9 Allylation of the C3 hydroxyl11 followed by
regioselective removal of the 5,6-isopropylidene group
delivered diol 13 in 84% yield. This diol was transformed
into the corresponding di-O-mesylate, from which the bis-
olefin 11a was obtained by treatment with NaI, in readiness
for a ring-closing metathesis. The NaI mediated conversion
of the intermediate di-O-mesylate to an olefin was initially
performed in diethyl ketone at 100 °C, which represents the
classical conditions for this type of reaction.12 However, the
presence of diethyl ketone caused the undesired formation
of up to 15% of 11b alongside 11a. The separation of 11b
required a chromatographic purification step. This trans-
acetalisation phenomenon was suppressed by switching to
N,N-dimethylacetamide (DMA) as the solvent, and a simple
distillation then became sufficient to provide analytically pure
11a. In addition, the yield of 11a in DMA (92%) was
superior to the same reaction performed in diethyl ketone,
even when taking into account the formation of 11b in the
mass balance. Ring closure metathesis (RCM) of 11a using
the Grubbs first generation catalyst8,13 gave the tricyclic olefin
10 in 85% yield. Purification of 10 could again be performed
by distillation. In their studies of the application of the
Grubbs RCM reaction to synthesize carbohydrates, van
Boom and co-workers14 reported a 63% yield for the
formation of 10 at a substrate concentration of 0.02 M in
order to minimize oligomerization. Independently, we had
performed the same cyclisation at a concentration of 0.05
M and a catalyst loading of 3.5 mol %. On a 30 g scale the
yield was consistently over 80%. At higher dilutions, the
yield was improved to over 90% but at the expense of
practicality. At concentrations higher than 0.05 M formation
of oligomers became significant and the yield decreased
significantly. Treatment of 10 with N-bromosuccinimide
(NBS) in aqueous THF followed by NaOH15 gave the
epoxide 14b. Subsequent opening with sodium azide in
2-methoxyethanol afforded the desired azide 15b regioisomer
in 48% yield (3 steps). Inversion of configuration was
achieved by oxidation with the Dess-Martin periodinane
reagent16 to 16, reduction with NaBH4, and methylation to
give the fully functionalized dihydropyran subunit 9 (Scheme
equivalent to malayamycin A in terms of fungicidal activity.
However, further exploration of the efficacy of this com-
pound was hampered by the supply of material, which could
be delivered by synthesis. This stood in contrast to malaya-
mycin A, which is supplied in sufficient amounts by
fermentation. Consequently, we set out to revise our first
generation approach to N-malayamycins. Herein we report
full details of a process allowing for the delivery of sufficient
amounts of material for the advanced evaluation of 1-cy-
tosinyl-N-malayamycin A (3) as a fungicide in crop protec-
tion.
Results and Discussion
Synthesis Plan. Our strategy was designed to rely upon
substrate-based stereocontrols only. To achieve this goal, the
retrosynthesis outlined in Scheme 1 was envisaged. Ring
closure metathesis8 of an appropriate diene 11a prepared from
commercially available9 1,2,5,6-di-O-isopropylidene-R-D-
allofuranose 12 would generate the unsaturated perhydro-
furopyran 10, which would undergo regio- and stereoselec-
tive functionalization to install the azido and methoxy groups
in a cis-relationship (9). An anomeric phenylthio group would
then be installed and used to introduce the cytosine en route
to the target molecule. The activation of thioglycosides with
thiophilic agents as a means to prepare bicyclic nucleosides
was previously exploited.2l,3c,10 A major challenge, however,
(10) For related methods, see (a) Knapp, S.; Shieh, W.-C. Tetrahedron Lett. 1991,
32, 3627. (b) Knapp, S.; Shieh, W.-C.; Jaramillo, C.; Trilles, R. V.; Nandan,
S. R. J. Org. Chem. 1994, 59, 946.
(11) Bhattacharjee, A.; Chattopadhyay, P.; Kundu, A. P.; Mukhopadhyay, R.;
Bhattacharjya, A. Indian J. Chem. 1996, 35B, 69.
(12) Jones J. K. N.; Thompson, J. L. Can. J. Chem. 1957, 35, 955.
(13) Schwab, P.; Grubbs, R. H.; Ziller, J. W. J. Am. Chem. Soc. 1996, 118, 100.
(14) Leeuwenburgh, M. A.; Kulker, C.; Duynstee, H. I.; Overkleeft, H. S.; van
der Marel, G. A.; van Boom, J. H. Tetrahedron 1999, 55, 8253.
(15) (a) Bannard, R. A. B.; Casselman, A. A.; Hawkins, L. R. Can. J. Chem.
1965, 43, 2398. (b) Bannard, R. A. B.; Casselman, A. A.; Langstaff, E. J.;
Moir, R. Y. Can. J. Chem. 1968, 46, 35.
(7) Hanessian, S.; Marcotte, S.; Huang, G.; Crowley, P. J.; Loiseleur, O. Patent
app. No. WO 2005005432, January 20, 2005.
(8) For pertinent reviews, see (a) Grubbs, R. H. Tetrahedron 2004, 60, 7117.
(b) Grubbs, R.; Chang, S. Tetrahedron 1998, 54, 4413. (c) Armstrong, S.
K. J. Chem. Soc., Perkin Trans. 1 1998, 371. (d) Fu¨rstner, A.; Picquet, M.;
Bruneau, C.; Dixneuf, P. H. Chem. Commun. 1998, 1315. (e) Schuster, M.;
Blechert, S. Angew. Chem., Int. Ed. Engl. 1997, 36, 2037.
(9) 1,2,5,6-Di-O-isopropylidene-R-D-allofuranose (12) is available at Carbop-
harm Gmbh, Industriestrasse 10, A-8502 Lannach, Austria (www.carbop-
harm.com).
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519

Scheme 2. Synthesis of the perhydrofuropyran 9
Scheme 3
2). Overall, this 11-step sequence was carried out on a 100
g scale and required only three purifications by chromatog-
raphy (one for the intermediate 15b and two for the
conversion of 16 to 9).
endo face of the bicyclic motif. This preference may be
rationalized by considering the favorable electron-donating
ability of the C-H σ bond with the developing antibonding
orbital in the transition state model TS-1 leading to the
observed epoxide.17 Approach of the peracid from the
opposite side as depicted in TS-2 would not benefit from
the same stereoelectronic effect. Likewise, treatment with
NBS in aqueous THF gave the endo bromonium ion, which
underwent trans-diaxial attack by a hydroxide ion leading
to the diaxial bromohydrin 17. Subsequent treatment with
base generated the correct regioisomeric epoxide 14b
(Scheme 3). Interestingly, dihydroxylation of 10 under
Upjohn conditions (OsO4, NMO)18 was nonselective, provid-
ing a 1:1 mixture of diols 18.
The syn-relationship of the azido and the methoxy groups
in 9 was readily confirmed by coupling constants in 1H NMR
spectroscopy data. In addition, the establishment of the
desired configuration at C6 and C7 was reinforced by the
observation of strong NOE enhancements between H7a and
H7 in 15b and H7a and both H7 and H6 in 9 (Figure 2).
Our initial efforts to functionalize the endocyclic double
bond in 10 relied on a direct epoxidation to 14b with mCPBA
followed by trans-diaxial ring opening with the azide ion
(Scheme 3). However, this protocol led to the regioisomeric
azide 15a.5,6 Evidently, epoxidation had occurred from the
Introduction of the Cytosine Moiety. The stage was now
set for the crucial introduction of the cytosine base. Removal
of the 2,3-O-isopropylidene protecting group was investi-
gated first. Attempts to deprotect model substrate 10 under
a variety of acidic conditions led to decomposition of the
constrained bicyclic dioxa core with concomitant disappear-
(16) (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155. (b) Dess, D.
B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277. For a safe preparation
of the reagent, see: (c) Frigerio, M.; Santagostino, M.; Sputore, S. J. Org.
Chem. 1999, 64, 4537.
(17) Cieplak, A. S. J. Am. Chem. Soc. 1981, 103, 4540.
(18) Van Rheenen, V.; Kelly, R. C.; Cha, D. Y. Tetrahedron Lett. 1976, 23,
1973.
Figure 2. NOE correlations (NOESY) and coupling constant
data for 15 and 9.
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Table 1. Removal of the ketal group in 9
Scheme 4. Stereocontrolled introduction of the pyrimidine
and completion of the synthesis
PhSH
(equiv) solvent
yield
(%)
entry
H+
temp
1
2
3
4
5
Amberlyst-15
Amberlyst-15
Amberlyst-15
Montmorillonite K10
Nafion NR 50
3
6
3
3
3
CH2Cl2 5 °C
CH2Cl2 5 °C
CH3OH 5 °C to rt
CH2Cl2 5 °C to rt
CH2Cl2 5 °C to rt
56a
84
0b
0
0
a Isolation of 31% of 21. b Formation of 22 (22%) and 23 (25%).
ance of the vinylic protons according to 1H NMR spectros-
copy. Consequently, we turned our attention to 9, in which
the double bond had been functionalized. Treatment with
Amberlyst-15 in MeOH cleanly afforded the mixed acetal
19 (Table 1). We then anticipated that the addition of
benzenethiol to this acid-mediated furanoside opening might
facilitate the cleavage of the acetal, while delivering directly
diphenyl dithioacetal 20, a key intermediate in our first
generation synthesis of N-malayamycins.6 Indeed, treatment
of 9 with 3 equiv of benzenethiol in the presence of
Amberlyst-15 suspended in CH2Cl2 delivered 20 in 56% yield
along with 31% of the cyclic acetal 21 (entry 1, Table 1).
Increasing the stoichiometry in benzenethiol to 6 equiv
afforded 20 in 84% yield (entry 2).
in the presence of N4-acetyl bis-O-TMS cytosine led to 25
in 96% yield as the only detectable anomer, as a result of
pivalate participation.10b,20 Reduction of the azido group in
25 under modified Staudinger conditions,21 followed by
treatment with trichloroacetyl isocyanate10b and deprotection
with methylamine gave 1-cytosinyl-N-malayamycin A 3, as
a colorless solid. The overall sequence could be easily
reproduced on a gram scale.
Conclusions
We have described a process for the preparation of
N-malayamycin analogue 3 that proceeds in 4.5% yield over
18 steps.22 The key carbocyclization step to the bicyclic
perhydrofuropyran intermediate involved a Grubbs ring
closure metathesis, which proceeded in excellent yield.
Regioselective functionalization of the bicyclic olefin was
achieved through stereocontrolled epoxidation and diaxial
opening with an azide ion. The formation of the bicyclic
The choice of Amberlyst to mediate the dithioacetal
formation was critical to its success. Indeed, use of Mont-
morillonite K10 or Nafion NR 50 did not promote the desired
conversion. Competition between PhSH and MeOH was a
problem when MeOH was used as the solvent instead of
CH2Cl2 and led to the isolation of the mixed acetals 22 and
23. Cyclization of 20 via an iodo sulfonium intermediate
triggered by N-iodosuccinimide (NIS) followed by the
ensuing thionium ion intermediate provided directly the
bicyclic phenylthio glycoside 24 in 70% yield, predominantly
as the R-anomer (Scheme 4).
(19) Veeneman, G. H.; van Leuwen, S. H.; van Boom, J. H. Tetrahedron Lett.
1990, 31, 1331.
(20) Sato, S.; Nunomura, S.; Nakano, T.; Ito, Y.; Ogawa, T. Tetrahedron Lett.
1988, 29, 4097.
(21) (a) Vaultier, M.; Knouzi, N.; Carrie, R. Tetrahedron Lett. 1983, 24, 763.
(b) Staudinger, H.; Meyer, J. HelV. Chim. Acta 1919, 2, 635.
(22) First generation route to 3: 25 steps, 2% yield (ref 6).
To ensure a stereocontrolled introduction of the cytosinyl
unit, we chose to protect the C2-hydroxyl as a pivalate (8).
Activation of the phenylthio group with NIS and triflic acid19
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521

N-nucleoside was achieved by activation of a thioglycoside,
proceeding sequentially via sulfonium and thionium inter-
mediates. The present route proved to be scalable and could
solve the supply problem, allowing an extensive exploration
of the efficacy of 3 as a fungicide for crop protection.
layer was extracted with hexane (500 mL × 2). The
combined organic extracts were washed with brine (500 mL
× 3), dried over MgSO4, and concentrated in vacuo. The
residue was purified by Kugelrohr distillation under reduced
pressure to afford 67 g of diene 11a (93%) as a colourless
oil. Rf 0.76 (SiO2, 1:1 hexane/AcOEt); 1H NMR (500 MHz,
CDCl3) δ 5.97-5.80 (m, 2H), 5.78 (d, 1H, J ) 4.2 Hz),
5.44 (dt, 1H, J ) 16.5, 1.2 Hz), 5.30 (dq, 1H, J ) 16.5, 1.0
Hz), 5.27 (dt, 1H, J ) 10.0 1.0 Hz), 5.22 (dq, 1H, J ) 10.0
1.2 Hz), 4.61 (t, 1H, J ) 4.0 Hz), 4.42 (dd, 1H, J ) 8.8, 7.0
Hz), 4.18 (ddt, 1H, J ) 13.5, 5.5, 1.0 Hz), 4.11 (ddt, 1H, J
) 13.5, 6.0, 1.0 Hz), 3.52 (dd, 1H, J ) 8.8, 4.0 Hz), 1.60
(s, 3H), 1.36 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 134.9,
134.4, 118.7, 118.1, 107.3, 103.7, 82.2, 79.0, 77.7, 71.7, 26.8,
26.5.
(2R,3R,3bR,7aR)-2,2-Dimethyl-3a,5,7a,8a-tetrahydro-
3bH-[1,3]dioxolo[4,5]furo[3,2-b]pyran (10). A solution of
11a (30 g, 133 mmol) and Grubbs first generation catalyst
(3.83 g, 4.65 mmol) in 2.6 L of CH2Cl2 saturated with argon
was refluxed under a slight flow of argon for 8 h. After
completion of the reaction as shown by TLC (SiO2, hexanes/
AcOEt, 3.1), the reaction mixture was washed with water
(600 mL), dried with MgSO4, and concentrated in vacuo.
Purification of the residue by filtration over a pad of SiO2
(elution with CH2Cl2) followed by Kugelrohr distillation
under reduced pressure afforded 22.4 g of 10 (85%) as a
pale brown solid. Rf 0.60 (SiO2, AcOEt); mp 64-65 °C;
[R]20D +17.7 (c 3.3 in CHCl3); 1H NMR (400 MHz, CDCl3)
δ 6.18 (d, 1H, J ) 8.1 Hz), 5.83 (d, 1H, J ) 3.5 Hz), 5.64
(d, 1H, J ) 2.1 Hz), 4.63 (t, 1H, J ) 3.6 Hz), 4.38 (s, 1H),
4.40 (s, 2 H), 3.26 (dd, 1H, J ) 8.1, 3.9 Hz), 1.53 (s, 3H),
1.32 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 127.3, 126.3,
113.2, 105.6, 78.9, 76.1, 69.4, 68.6, 26.0, 25.8; MS (ESI)
199.1 [M + H]+.
(2R,3R,3bR,6R,7R,7aR)-7-Azido-2,2-dimethyl-hexahy-
dro-[1,3]dioxolo[4,5]furo[3,2-b]pyran-6-ol (15b). To a
solution of 10 (45.6 g, 230 mmol) in THF-H2O (1:1 v/v,
600 mL) cooled by ice bath was added NBS (51.0 g, 286
mmol). After 10 min, the ice bath was removed and the
reaction mixture was stirred at room temperature for 2 h.
The mixture was quenched with aq. satd. Na2S2O3 (380 mL)
and stirred for 20 min. The phases were separated, and the
aqueous layer was extracted with EtOAc (300 mL × 3). The
combined organic extracts were dried over MgSO4, filtrated,
and concentrated in vacuo. The resulting brown crystalline
solid was engaged in the next reaction. Rf 0.52 (SiO2,
AcOEt).
A solution of the above residue in THF (550 mL) was
treated with aq. 1 N NaOH (550 mL) and refluxed for 1 h.
The mixture was then poured into H2O (300 mL) and
extracted with EtOAc (300 mL × 2). The combined organic
layer was washed with brine (150 mL), dried over MgSO4,
and concentrated in vacuo to give 48.8 g of the epoxide 14b
as a yellowish crystalline solid, which did not require further
purification and which was engaged in the next reaction. Rf
0.46 (SiO2, AcOEt).
Experimental Section
(R)-1-[(3aR,5R,6R,6aR)-6-(Allyloxy)-tetrahydro-2,2-
dimethylfuro[2,3-d][1,3]dioxol-5-yl]ethane-1,2-diol (13). In
a reactor equipped with a mechanical stirrer, a solution of
12 (100 g, 384 mmol) in 1.6 L of CH2Cl2 was treated
sequentially with allyl bromide (50 mL, 591 mmol), aqueous
NaOH (50%, 1.6 L), and tetrabutylammonium iodide (14.2
g, 38.4 mmol). After stirring vigorously at 23 °C for 5 h,
the aqueous layer was extracted with tert-butylmethyl ether
(TBME) (500 mL × 2). The organic extracts were combined,
washed with aq. satd. NH4Cl (1 L + addition of solid NH4-
Cl for neutralization of NaOH), dried over MgSO4, and
concentrated in vacuo. Purification of the residue by recrys-
tallization in hexane afforded 106.3 g (92%) of the desired
allyl ether as colorless crystals, which were engaged in the
next reaction. Rf 0.51 (SiO2, AcOEt); mp 36.0-37.5 °C.
A solution of the above material (106 g, 353 mmol) in
AcOH-H2O 75:25 v/v (700 mL) was kept at 23 °C for 15
h and then concentrated in vacuo at 30 °C. TBME (300 mL)
and aq. satd. NaHCO3 (30 mL) were added to the residue.
The aqueous layer was extracted with TBME (200 mL ×
3). The organic extracts were combined, washed with brine
(30 mL), dried over MgSO4, and concentrated in vacuo to
give 84 g (91%) of the diol 13 as a colorless solid, which
did not require further purification. Rf 0.11 (SiO2, AcOEt);
1H NMR (500 MHz, CDCl3) δ 6.01-5.92 (m, 1 H), 5.79
(d, 1H, J ) 4.0 Hz), 5.34 (dq, 1H, J ) 18.5, 1.0 Hz), 5.27
(dd, 1H, J ) 10.0, 1.0 Hz), 4.64 (t, 1H, J ) 4.0 Hz), 4.26
(ddt, 1H, J ) 12.0, 5.3, 1.0 Hz), 4.12-4.02 (m, 3H), 3.93
(dd, 1H, J ) 9.0, 4.0 Hz), 3.74 (dd, 1H, J ) 11.0, 5.0 Hz),
3.70 (dd, 1H, J ) 11.0, 4.5 Hz), 2.40-1.80 (br s, 2H), 1.58
(s, 3H), 1.37 (s, 3H).
(3aR,5R,6R,6aR)-6-(Allyloxy)-tetrahydro-2,2-dimethyl-
5-vinylfuro[2,3-d][1,3]dioxole (11a). To a solution of 13
(83 g, 319 mmol) and triethylamine (111 mL, 798 mmol) in
400 mL of CH2Cl2 at 0 °C under an atmosphere of argon
was added dropwise methanesulfonyl chloride (54 mL, 702
mmol) (strongly exothermic). After 30 min at 0 °C, water
(200 mL) was added and the phases were separated. The
aqueous layer was extracted with CH2Cl2 (200 mL × 2).
The organic extracts were combined, washed with aq. satd.
NaHCO3 (50 mL), dried over MgSO4, and concentrated in
vacuo to give the bis-mesylate as a pale yellowish oil, which
was engaged in the next reaction. Rf 0.33 (SiO2, 1:1 hexane/
AcOEt).
A solution of the above material in 1.5 L of N,N-
dimethylacetamide under an atmosphere of argon was treated
with NaI (243 g, 1.59 mol). The resulting suspension was
stirred at 100 °C for 2.5 h, whereas a dark-brown coloration
developed gradually. The reaction mixture cooled to room
temperature was diluted with water (1 L) and treated with
aq. satd. Na2S2O3 (1.5 L). After stirring for 1 h, hexane (1
L) was added. The phases were separated, and the aqueous
The above epoxide 14b was dissolved in 2-methoxyetha-
nol (500 mL), and sodium azide (17.9 g, 276 mmol) was
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added. The mixture was heated at 130 °C for 2 h, diluted
with brine (300 mL) and water (100 mL), and extracted with
EtOAc (300 mL × 3). The combined organic layer was dried
over MgSO4 and concentrated in vacuo. The residue was
purified by flash silica gel chromatography (cyclohexane/
EtOAc 4:1 to 2:1, gradient) to afford 28.5 g of azide 15b
(48%) as a pale yellowish crystalline solid. Rf 0.19 (SiO2,
1:1 cyclohexane/AcOEt); mp 115.0-117.0 °C; [R]20D +42.5
(c 2 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.84 (d, 1H,
J ) 3.4 Hz), 4.66 (t, 1H, J ) 3.6 Hz), 4.34 (t, 1H, J ) 2.3
Hz), 4.31 (dd, 1H, J ) 9.8, 2.3 Hz), 3.90-3.77 (two m, 3
H), 3.54 (dd, 1H, J ) 9.8, 4.2 Hz), 2.30 (d, 1H, J ) 6.8
Hz), 1.58 (s, 3H), 1.35 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 113.8, 104.3, 76.3, 75.0, 72.0, 69.7, 68.6, 68.5, 26.1, 25.9;
LRMS (ESI): 258.1 [M + H]+.
(2R,3R,3bR,6S,7R,7aR)-7-Azido-6-methoxy-2,2-dimeth-
yl-hexahydro-[1,3]dioxolo[4,5]furo[3,2-b]pyran (9). To a
solution of 15b (28.5 g, 111 mmol) in CH2Cl2 (500 mL) at
water bath controlled room temperature was added Dess-
Martin periodinane (56.5 g, 133 mmol). The mixture was
stirred for 2.5 h. Aq. satd. NaHCO3 (200 mL) and Na2S2O3
(200 mL) were then added sequentially. The mixture was
stirred for 20 min, and the phases were separated. The
aqueous layer was extracted with CH2Cl2. The combined
organic layer was washed with water (300 mL), dried over
MgSO4, and concentrated in vacuo to give 20.1 g of ketone
16 as a yellowish resin, which was engaged in the next
reaction. Rf 0.38 (SiO2, 75:25 cyclohexane/AcOEt).
1.58 (s, 3H), 1.35 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
107.3, 105.8, 76.2, 76.1, 74.8, 73.6, 66.1, 59.0, 57.5, 26.3,
26.0; MS (ESI) 244 [M + H - N2]+.
(2R,3R,4S,5S)-4-Azido-tetrahydro-2-[(R)-1-hydroxy-
2,2-bis(phenylthio)ethyl]-5-methoxy-2H-pyran-3-ol (20).
To a solution of 9 (16.5 g, 60.8 mmol) in CH2Cl2 (400 mL)
at 5 °C, PhSH (37.3 mL, 365 mmol) was added followed
by Amberlyst-15 (16.5 g). The mixture was stirred at 5 °C
for 4 h, then filtrated, quenched with aq. satd. NaHCO3 (200
mL), and stirred for 15 min. After the separation of the two
phases, the aqueous layer was extracted with CH2Cl2 (60
mL × 2). The combined organic layers were washed with
brine (300 mL), dried with MgSO4, filtrated, and concen-
trated in vacuo. Purification of the crude product by flash
silica gel chromatography (cyclohexane/EtOAc, 9:1 to 7:3,
gradient) afforded 22.1 g of dithioacetal 20 as a colorless
oil (84%), which crystallized upon addition of seed crystals.
Rf 0.46 (SiO2, Et2O); mp 93.0-94.5 °C; [R]20D +28.3 (c 0.79
1
in CHCl3); H NMR (400 MHz, CDCl3) δ 7.50-7.26 (m,
10H), 4.88 (d, 1H, J ) 2.2 Hz), 4.26 (br t, 1H), 3.91 (dd,
1H, J ) 8.2, 2.2 Hz), 3.76 (t, 1H, J ) 9.1 Hz), 3.69 (dd,
1H, J ) 9.1, 3.6 Hz), 3.63 (ddd, 1H, J ) 10.6, 4.8, 1.1 Hz),
3.41 (s, 3H), 3.34 (ddd, 1H, J ) 10.6, 4.8, 3.2 Hz), 3.22 (t,
1H, J ) 10.6 Hz); 13C NMR (100 MHz, CDCl3) δ 132.3,
129.1, 128.9, 128.0, 127.9, 76.0, 75.6, 72.7, 71.9, 63.2, 62.4,
56.9; HRMS (FAB) calcd for C20H23N3O4S2 [M+] 433.1130,
found 433.1132.
(2R,3R,3aS,6S,7R,7aR)-7-Azido-hexahydro-6-methoxy-
2-(phenylthio)-2H-furo[3,2-b]pyran-3-ol (24). To a solution
of 20 (13.8 g, 31.8 mmol) in dry CH2Cl2 (250 mL) at 0 °C
and under an atmosphere of argon, NIS (7.52 g, 33.4 mmol)
was added. After stirring at room temperature for 20 min,
the mixture was quenched by adding aq. satd. Na2S2O3 (300
mL) and then stirred for 10 min. Brine (200 mL) was added,
and the two phases were separated. The aqueous layer was
extracted with CH2Cl2 (100 mL × 2). The combined organic
extracts were washed with brine (200 mL), dried with
MgSO4, filtrated, and concentrated in vacuo. Flash silica gel
chromatography (cyclohexane/EtOAc 8:2 to 6:4, gradient)
afforded 6.0 g of the R-anomer of phenylthio glycoside 24
(58%) as a colourless crystalline solid and 1.2 g of the
â-anomer (12%) as a resin. At this stage, the two anomers
of 24 could be combined and converted to the 1-cytosinyl-
N-nucleoside 25 or converted separately to 25. R-Anomer:
To a solution of the above ketone 16 in MeOH (400 mL)
at 0 °C was added NaBH4 (4.46 g, 118 mmol). The resulting
mixture was stirred at room temperature for 1 h and then
concentrated in vacuo without heating. The residue was taken
up with EtOAc (400 mL) and washed with brine (400 mL).
The aqueous layer was extracted with EtOAc (350 mL ×
2). The organic extracts were combined, dried over MgSO4,
and concentrated in vacuo. Purification of the residue by flash
silica gel chromatography (cyclohexane/EtOAc 4:1 to 2:1,
gradient) afforded 16.4 g of the desired alcohol as a
colourless oil, which was engaged in the next reaction. Rf
0.42 (SiO2, 75:25 cyclohexane/AcOEt); mp 113.0-115.0 °C.
To a solution of the above alcohol in THF (200 mL) at 0
°C under an atmosphere of argon was added NaH (60%
suspension, 5.08 g, 127 mmol). After stirring for 1 h at 0
°C, MeI (7.89 mL, 127 mmol) was added dropwise. The
reaction mixture was stirred at room temperature for 1.5 h
and was quenched by adding satd. NH4Cl (300 mL). The
mixture was extracted with EtOAc (250 mL × 3). The
combined organic layer was washed with brine (150 mL),
dried over MgSO4, and concentrated in vacuo. The residue
was purified by flash silica gel chromatography (cyclohex-
ane/EtOAc, 6:1 to 4:1, gradient) to afford 9 (16.9 g, 56%)
as a colorless oil, which crystallized upon conservation at 4
°C. Rf 0.41 (SiO2, 1:1 cyclohexane/AcOEt); mp 90.5-92.5
Rf 0.22 (SiO2, 80:20 Et2O/cyclohexane); mp 131.0-132.5
1
°C; [R]20 +87.5 (c 0.76 in CHCl3); H NMR (400 MHz,
D
CDCl3) δ 7.59-7.53 (m, 4H), 7.38-7.30 (m, 6H), 5.30 (s,
1H), 4.58 (br t, 1H), 4.34 (d, 1H, J ) 4.8 Hz), 3.91 (dd, 1H,
J ) 10.5, 4.8 Hz), 3.88 (dd, 1H, J ) 10.0, 2.5 Hz), 3.62
(dd, 1H, J ) 10.0, 4.8 Hz), 3.58 (t, 1H, J ) 10.5 Hz), 3.46
(ddd, 1H, J ) 10.5, 4.8, 3.5 Hz), 3.43 (s, 3H), 2.35 (br s,
1H, OH); 2.50 (d, 1H, J ) 2.2 Hz); 13C NMR (100 MHz,
CDCl3) δ 134.4, 130.9, 128.9, 127.1, 93.8, 76.2, 74.1, 73.9,
69.5, 65.6, 58.8, 57.4; HRMS (FAB) calcd for C14H18N3O4S
[M + H]+ 324.1018, found 324.1008. â-Anomer: Rf 0.12
(SiO2, 80:20 Et2O/cyclohexane).
1
°C; H NMR (500 MHz, CDCl3) δ 5.85 (d, 1H, J ) 4.0
Hz), 4.62 (t, 1H, J ) 4.0 Hz), 4. 58 (t, 1H, J ) 3.2 Hz),
3.98 (dd, 1H, J ) 10.5, 4.5 Hz), 3.86 (dd, 1H, J ) 9.5, 3.2
Hz), 3.65 (dd, 1H, J ) 9.5, 4.0 Hz), 3.62 (t, 1H, J ) 10.5
Hz), 3.58 (ddd, 1H, J ) 10.5, 4.5, 3.2 Hz), 3.45 (s, 3H);
(2R,3R,3aR,6S,7R,7aR)-7-Azido-hexahydro-6-methoxy-
2-(phenylthio)-2H-furo[3,2-b]pyran-3-yl Pivalate (8). To
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523

the alcohol 24 (1.68 g, 5.2 mmol) in pyridine (30 mL),
DMAP (3.18 g, 26.0 mmol) and pivaloyl chloride (1.60 mL,
13.0 mmol) were added and the mixture was stirred at room
temperature for 1.5 h. Aq. satd. NaHCO3 (100 mL) and
TBME (100 mL) were added, and the two phases were
separated. The aqueous layer was extracted with TBME (100
mL × 2). The combined organic extracts were washed with
0.1 N HCl (100 mL × 2) and aq. satd. NaHCO3 (100 mL),
dried over MgSO4, and concentrated in vacuo. Purification
of the crude residue by flash silica gel chromatography
(cyclohexane/EtOAc 95:5 to 92:8, gradient) afforded 2.11 g
of pivalate 8 as a colourless resin (quantitative). R-Anomer:
154.3, 143.8, 96.8, 91.1, 76.6, 75.9, 72.3, 71.6, 65.8, 58.8,
57.6, 38.7, 29.4, 26.9; LC-MS m/z (relative intensity): 450
(M+, 100).
The â-anomer of 8 was converted to 25 in 52% yield
under identical experimental conditions.
(2R,3R,3aS,6S,7R,7aR)-[2-(4′-Amino-2′-oxo-2H-pyrimi-
din-1′-yl)-3-hydroxy-6-methoxy-hexahydrofuro[3,2-b]py-
ran-7-yl]-urea (3) (1-Cytosinyl-N-malayamycin A). To a
solution of azide 25 (450 mg, 1.0 mmol) in anhydrous THF
(30 mL) saturated with argon was added Me3P (2.50 mL, 1
M solution in toluene, 2.5 mmol) at room temperature. After
stirring for 30 min, water (150 µL) was added. The resulting
mixture was refluxed for 40 min and then concentrated. The
residue was dried under reduced pressure (0.1 mmHg) for 2
h and dissolved in dry CH2Cl2 (20 mL). To this solution
was added trichloroacetyl isocyanate (480 µL, 4.0 mmol) at
room temperature under argon atmosphere. After stirring for
1 h, CH2Cl2 was removed and the residue was dissolved in
MeOH (25 mL) and 40% MeNH2 in H2O (25 mL) and stirred
at room temperature over 3 days. Evaporation of the mixture
and purification by flash silica gel chromatography (CH2-
Cl2/MeOH/25% aq. NH4OH, 100:15:1.5 to 100:33:3.3,
gradient) afforded 154 mg of 1-cytosinyl-N-malayamycin A
3, as a pale colourless solid (45%). Rf 0.32 (SiO2, 70:30:3
Rf 0.37 (SiO2, 8:2 cyclohexane/AcOEt); [R]20 +187.3 (c
D
1
0.41 in CHCl3); H NMR (400 MHz, CDCl3) δ 7.51-7.27
(m, 5H), 5.90 (d, 1H, J ) 3.4 Hz), 5.68 (t, 1H, J ) 3.4 Hz),
4.62 (t, 1H, J ) 3.2 Hz), 3.97 (dd, 1H, J ) 10.0, 3.4 Hz),
3.92 (dd, 1H, J ) 11.6, 4.4 Hz), 3.84 (dd, 1H, J ) 10.0, 3.4
Hz), 3.61 (dd, 1H, J ) 11.6, 10.0 Hz), 3.51 (m, 1H), 3.47
(s, 3H), 1.30 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 177.1,
134.8, 131.6, 129.5, 127.8, 92.3, 76.8, 75.3, 73.9, 70.4, 66.0,
59.2, 57.9, 39.8, 27.6; HR-MS (FAB) calcd for C19H26N3O5S
[M + H]+ 408.1601, found 408.1586.
The â-anomer of 24 was converted to the â-anomer of 8
in 85% yield under identical experimental conditions.
(2R,3R,3aR,6S,7R,7aR)-7-Azido-2-(4′-acetylamino-2′-
oxo-3′,4′-dihydro-2H-pyrimidin-1′-yl)-6-methoxy-3-piv-
aloyloxy-hexahydrofuro[3,2-b]pyran (25). To a solution of
8 (1.22 g, 3.0 mmol), N-4-acetyl bis-O-TMS cytosine (2.14
g, 7.2 mmol), and NIS (1.62 g, 7.2 mmol) in CH2Cl2 (60
mL) at room temperature TfOH (625 µL, 7.2 mmol) was
added over a period of 15 min. After stirring for 6 h,
additional portions of NIS (0.68 g, 3.0 mmol) and TfOH
(260 µL, 3.0 mmol) were added, and the mixture was stirred
for 6 h. The mixture was quenched by adding a saturated
solution of Na2S2O3 (100 mL) and then stirred for 15 min.
After filtration over a pad of Hyflo, the two phases were
separated. The aqueous layer was extracted with CH2Cl2 (50
mL × 2). The combined organic extracts were washed with
aq. satd. NaHCO3 (100 mL) and dried with MgSO4. After
concentration in vacuo, the crude product was purified by
flash silica gel chromatography (EtOAc/MeOH, 100:1 to 99:
1, gradient) to afford 1.29 g of 25 as colourless resin (96%).
CH2Cl2/MeOH/25% aq. NH4OH); [R]20 +91.7 (c 0.06 in
D
1
MeOH); H NMR (400 MHz, CD3OD) δ 7.73 (d, 1H, J )
7.5 Hz), 5.87 (d, 1H, J ) 7.5 Hz), 5.67 (s, 1H), 4.97 (m,
1H), 4.19 (d, 1H, J ) 4.3 Hz), 4.05 (dd, 1H, J ) 10.6, 2.3
Hz), 3.96 (dd, 1H, J ) 11.6, 5.4 Hz), 3.66 (dd, 1H, J )
10.6, 5.3 Hz), 3.50 (m, 1H), 3.37 (s, 3H), 3.35 (m, 1H); 1H
NMR (500 MHz, D2O) δ 7.56 (d, 1H, J ) 7.5 Hz), 6.00 (d,
1H, J ) 7.5 Hz), 5.73 (s, 1H), 4.96 (br s, 1H), 4.33 (d, 1H,
J ) 5.3 Hz), 4.10-4.02 (m, 2H), 3.86-3.79 (m, 1H), 3.65
(dd, 1H, J ) 10.5, 5.0 Hz), 3.51 (app. t, 1H, J ) 11.2 Hz),
3.39 (s, 3H); 13C NMR (100 MHz, CD3OD) δ 166.7, 161.3,
156.9, 140.7, 94.8, 94.7, 76.9, 74.1, 73.5, 72.6, 66.6, 55.8,
48.8; FAB-MS m/z (relative intensity) 364 (M + Na+, 35),
342 (M + H+, 18), 325 (M+ - NH2, 8).
Acknowledgment
The experimental work of T. Vifian, M. Hellstern, and
R. Eckes is gratefully acknowledged. We thank the Analyti-
cal Department of Syngenta Crop Protection Basel for their
support. The project was made possible thanks to the strong
support and commitment of A. de Mesmaeker (Head
Research Chemistry).
Rf 0.53 (SiO2, 9:1 AcOEt/MeOH); [R]20 +98.8 (c 0.8 in
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ 10.1 (br s, 1H), 8.15
(d, 1H, J ) 7.5 Hz), 7.48 (d, 1H, J ) 7.5 Hz), 5.99 (s, 1H),
5.32 (d, 1H, J ) 4.5 Hz), 4.70 (br t, 1H), 3.95 (dd, 1H, J )
10.6, 4.3 Hz), 3.87 (dd, 1H, J ) 10.0, 2.8 Hz), 3.77 (dd,
1H, J ) 10.0, 4.5 Hz), 3.58 (t, 1H, J ) 10.6 Hz), 3.56 (ddd,
1H, J ) 10.6, 4.3, 2.5 Hz), 3.46 (s, 3H), 2.25 (s, 3H), 1.21
(s, 9H); 13C NMR (100 MHz, CDCl3) δ 178.2, 170.9, 162.9,
Received for review February 2, 2006.
OP0600299
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