Synthetic studies on amipurimycin: Total synthesis of a thymine nucleoside analogue

Article abstract of DOI:10.1021/jo8004815

(Chemical Equation Presented) Amipurimycin, a member of the complex peptidyl nucleoside family of antibiotics, is a Streptomyces-derived potent antifungal agent. The mechanism of action of amipurimycin, however, remains undetermined. Additionally, there are no reports on the total synthesis or structure-activity relationships (SAR) of this potentially useful bioactive compound. In a study aimed at the total synthesis and SAR studies of this natural product, the present research reports the development of a synthetic route to the central pyranosyl amino acid core of amipurimycin and its further elaboration, culminating in the synthesis of a unique thymine analogue. Utilizing a D-serine-derived dihydroaminopyrone as a strategic building block, the synthesis involves de novo construction of the fully functionalized C-3-branched carbohydrate amino acid core, followed by glycosidic attachment of thymine at C-1, and peptidic linking of the C-6 amine with the 1,2-aminocyclopentane carboxylic acid side chain.

Full text of DOI:10.1021/jo8004815

Synthetic Studies on Amipurimycin: Total Synthesis of a Thymine
Nucleoside Analogue
Christina S. Stauffer† and Apurba Datta*
Department of Medicinal Chemistry, The UniVersity of Kansas, 1251 Wescoe Hall DriVe,
Lawrence, Kansas 66045
adutta@ku.edu
ReceiVed February 29, 2008
Amipurimycin, a member of the complex peptidyl nucleoside family of antibiotics, is a Streptomyces-
derived potent antifungal agent. The mechanism of action of amipurimycin, however, remains
undetermined. Additionally, there are no reports on the total synthesis or structure-activity relationships
(SAR) of this potentially useful bioactive compound. In a study aimed at the total synthesis and SAR
studies of this natural product, the present research reports the development of a synthetic route to the
central pyranosyl amino acid core of amipurimycin and its further elaboration, culminating in the synthesis
of a unique thymine analogue. Utilizing a D-serine-derived dihydroaminopyrone as a strategic building
block, the synthesis involves de novo construction of the fully functionalized C-3-branched carbohydrate
amino acid core, followed by glycosidic attachment of thymine at C-1, and peptidic linking of the C-6
amine with the 1,2-aminocyclopentane carboxylic acid side chain.
Introduction
mg/kg (intravenous), and 10-30 mg/kg (oral), respectively.1
The mechanism of antifungal action of amipurimycin, however,
remains unknown.
The proposed amipurimycin structure was determined with
the help of extensive spectroscopic and chemical degradation
studies. Thus, amipurimycin was found to be made up of an
unusual C-3′ branched pyranose amino acid, appended to an
N-terminal amino acid residue at C-6′, and a glycosidic purine
nucleobase (Figure 1).2
However, the stereochemistry at C-6′ and the absolute
configurations at C-2′′/C-3′′ for the cis-aminocyclopentane
carboxylic acid derived side chain have not yet been ascertained.
Interestingly, the amipurimycin aminocyclopentane carboxylic
acid side chain fragment, also known as cispentacin, is itself
an antibiotic.3 Isolated from Streptomyces setonii, the enan-
tiopure natural cis-(1R,2S)-cispentacin was found to show good
antifungal activity against Candida albicans.3 Although a few
The complex peptidyl nucleoside antibiotic amipurimycin (1,
Figure 1) was isolated from Streptomyces noVoguineensis sp.
nov., a new strain of Streptomyces that was discovered in soil
samples collected from New Guinea during the 1970s.1 Ami-
purimycin displays impressive antifungal activity against several
phytopathogenic fungi, such as Pyricularia oryzae, Alternaria
kikuchiana, and Helminthosporium sigmoideum var. irregulare.1
Thus, in field tests, amipurimycin showed excellent controlling
and curative effect against blast disease of rice plants. In limited
toxicity studies, no toxicity to killifish was observed on exposure
to amipurimycin (10 ppm) after the first 2 days; however, all
the fish tested died on the third day. In mice and rats, the LD50
values of amipurimycin were found to be in the ranges 1-5
* To whom correspondence should be addressed. Phone: +1-785-864-4117.
Fax: +1-785-864-5326.
† Present address: Department of Chemistry, University of North Carolina,
Chapel Hill.
(1) (a) Iwasa, T.; Kishi, T.; Matsuura, K.; Wakae, O. J. Antibiot. 1977, 30,
1–10. (b) Harada, S.; Kishi, T. J. Antibiot. 1977, 30, 11–16.
(2) Goto, T.; Toya, Y.; Ohgi, T.; Kondo, T. Tetrahedron Lett. 1982, 23, 1271–
1274.
4166 J. Org. Chem. 2008, 73, 4166–4174
10.1021/jo8004815 CCC: $40.75 2008 American Chemical Society
Published on Web 05/09/2008

Synthetic Studies on Amipurimycin
FIGURE 1. Antifungal antibiotic amipurimycin.
syntheses of the various structural fragments of amipurimycin
have been reported,4,5 the total synthesis or structure-activity
relationship (SAR) studies of this natural product are yet to be
accomplished.
The demonstrated antifungal activity, challenging and partially
unresolved structural features, an as yet undetermined mecha-
nism of action, and limited synthetic or medicinal chemical
studies render amipurimycin an attractive target for exploration
as a new antifungal agent of potential utility. Accordingly, in
continuation of our interest in the peptide nucleoside antibiotics,6
we initiated a total synthesis of amipurimycin. The preliminary
results of our studies are described herein.
Results and Discussion
FIGURE 2. Retrosynthetic strategy and approach.
In the few reported syntheses of the amipurimycin structural
fragments, the studies were mostly focused on construction of
the hexopyranoside amino acid core of this natural product.4,5
The majority of the above methods employed carbohydrate
starting materials to construct the required C-3 (carbohydrate
numbering) branched pyranose framework, followed by sub-
sequent functional group manipulations to introduce the C-6
side chain amino acid functionality. Thus, in independent studies,
the research groups of Czernecki, Rauter, and Yoshimura have
utilized D-glucose as a starting material in their synthetic efforts
on amipurimycin.4 In contrast, in an interesting noncarbohy-
drate-based approach, a stereocontrolled cyclocondensation
between a serine-derived homochiral oxazolidine aldehyde
(Garner’s aldehyde) and an electron-rich polyoxygenated diene
was utilized by Garner to assemble the branched sugar amino
acid component of amipurimycin.5
It is evident from the available literature that, utilization of
carbohydrate starting materials in the synthesis of the sugar cores
of amipurimycin and other related peptidyl nucleoside antibiotics
has considerable merit when it can be applied in an efficient
manner.7 However, when the target end product incorporates
unusual residues or unnatural stereochemistry, the carbohydrate-
based approach often suffers from lengthy reaction sequences
and necessitates extensive protection-deprotection of functional
groups. In the present research, we therefore explored an
alternative synthetic strategy involving stereoselective de novo
construction of the nucleoside amino acid core of these targets,
starting from a structurally simpler and more flexible noncar-
bohydrate synthon.
In our retrosynthetic strategy, we envisaged initial construc-
tion of a fully functionalized pyranosyl amino acid core 2
(Figure 2), as present in amipurimycin, to be followed by its
glycosidic attachment at C-1 with the appropriate nucleobase,
and peptidic linking of the C-6 amine with cispentacin. For the
sake of simplicity, in our present synthesis, we decided to install
an S-configured natural amino acid functionality at C-6, the same
stereochemical configuration that is present in the other members
of the peptidyl nucleoside family of antibiotics.7 Similarly, the
naturally occurring and commercially available enantiopure
(1R,2S)-cispentacin was selected as the side chain amino acid
donor. The pivotal C-3 branched polyoxygenated carbohydrate
amino acid fragment 2 can be synthesized by appropriate
functional group transformations of the strategically function-
alized pyrone 5, which in turn can be obtained from the
enantiopure dihydropyran-2-one 6. Starting from D-serine, an
efficient synthetic route to the enantiopure aminopyrone 6 has
already been developed during our previously reported studies
on complex peptidyl nucleoside antibiotics.6a
As and when desired, appropriate modifications of the above
synthetic strategy can also lead to the other possible antipodes
at the above stereocenters.8
(3) (a) Iwamoto, T.; Tsujii, E.; Ezaki, M.; Fujie, A.; Hashimoto, S.; Okuhara,
M.; Kohsaka, M.; Imanaka, H.; Kawabata, K. J. Antibiot. 1990, 43, 1–7. (b)
Kawabata, K.; Inamoto, Y.; Sakane, K.; Iwamoto, T.; Hashimoto, S. J. Antibiot.
1990, 43, 513–518.
(4) (a) Hara, K.; Fujimoto, H.; Sato, K. I.; Hashimoto, H.; Yoshimura, J.
Carbohydr. Res. 1987, 159, 65–79. (b) Rauter, A. P.; Fernandes, A. C.; Czernecki,
S.; Valery, J.-M. J. Org. Chem. 1996, 61, 3594–3598. (c) Czernecki, S.; Valery,
J.-M.; Wilkens, R. Bull. Chem. Soc. Jpn. 1996, 69, 1347–1351. (d) Czernecki,
S.; Franco, S.; Valery, J.-M. J. Org. Chem. 1997, 62, 4845–4847.
(5) Garner, P.; Yoo, J. U.; Sarabu, R.; Kennedy, V. O.; Youngs, W. J.
Tetrahedron 1998, 54, 9303–9316.
(7) For reviews on complex peptidyl nucleoside antibiotics, see: (a) Garner,
P. Synthetic approaches to complex nucleoside antibiotics. In Studies in Natural
Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, The Nether-
lands, 1988; Stereoselective synthesis (Part A), Vol. 1, pp 397-435. (b) Isono,
K. Pharmacol. Ther. 1991, 52, 269–286. (c) Knapp, S. Chem. ReV. 1995, 95,
1859–1876. (d) Zhang, D.; Miller, M. J. Curr. Pharm. Des. 19995, 73–99, and
references therein.
(8) For example, starting from L-serine, the key syn-1,2-amino alcohol
fragment as required for the synthesis of the corresponding C6 antipode of the
lactone 6 can be easily obtained following a previously reported protocol from
our group: Ravi Kumar, J. S.; Datta, A. Tetrahedron Lett. 1999, 40, 1381–1384.
(6) For recent reports, see: (a) Bhaket, P.; Stauffer, C. S.; Datta, A. J. Org.
Chem. 2004, 69, 8594–8601. (b) Khalaf, J. K.; Datta, A. J. Org. Chem. 2005,
70, 6937–6940. (c) Stauffer, C. S.; Bhaket, P.; Fothergill, A. W.; Rinaldi, M. G.;
Datta, A. J. Org. Chem. 2007, 72, 9991–9997.
J. Org. Chem. Vol. 73, No. 11, 2008 4167

Stauffer and Datta
SCHEME 1
In accordance with the above strategy, and employing a
recently developed protocol from our laboratory,6a readily
available D-serine was converted to the corresponding strategi-
cally functionalized aminobutenolide 6 (Scheme 1) in good
overall yield (61%, seven steps). Subsequent osmium tetroxide-
catalyzed dihyroxylation of 6 under reported conditions resulted
in the known diol 7 with high stereocontrol.6a Acetonide
protection of the diol functionality provided the corresponding
fully protected pyrone derivative 8 in near-quantitative yield.
DIBAL mediated partial reduction of the lactone 8 to the
corresponding lactol, followed by anomeric O-alkylation with
4-methoxybenzyl bromide (PMB-Br) yielded the ꢀ-glycosidic
derivative 9 as the only product. The highly stereoselective
formation of the ꢀ-anomer can be attributed to the sterically
hindered C-2/C-3 substituents influencing the product formation
from the less hindered face. One-pot hydrolytic cleavage of both
of the acetonide protecting groups generated the trihydroxy
derivative 10. In the 1H NMR spectrum of 10, the characteristic
trans-coupling constant as observed for the anomeric proton (J1,2
) 8.0 Hz) helped confirm the assigned C-1 stereochemistry.
Selective silyl protection of the resulting primary hydroxyl group
led to the C-2/C-3 free diol 11 in good yield. Our next goal
was selective C-3 oxidation of the above diol 11 toward
construction of the branched polyhydroxylated C-3 side chain
as present in amipurimycin. Accordingly, exploiting the dif-
ferential reactivity profile between the C-2 and C-3 hydroxyl
functionalities, reaction of the diol 11 with 1.1 equiv of Ac2O
under carefully controlled conditions resulted in the correspond-
ing monoacetylated deivative 12. Expectedly, in the above
reaction, the more reactive equatorial C-2 hydroxyl group
underwent preferential reaction over the axial hydroxyl group
at C-3.9 The free hydroxyl group at C-3 was then converted to
the ketone 13. Subsequent Wittig olefination of 13 with
(carbomethoxymethylene)triphenylphosphorane resulted in the
selective formation of the corresponding E-olefin 14. High-
resolution NMR studies (1H, 13C, COSY, NOE) confirmed the
olefin geometry and the assigned structure of 14. As supported
by literature precedence,4b the trans-selectivity in the above
E-alkylidene formation is probably attributable to the steric
influence of the neighboring C-2 acetoxy substituent.
Reduction of the two ester functionalities of 14, in the
presence of excess DIBAL, followed by acetylation of the
resulting diol with acetic anhydride formed the diacetate
derivative 15 in good overall yield. With the aim to form
the C-6 amino acid functionality, the silyl protecting group of
15 was removed to unmask the primary alcohol 16. Subsequent
oxidation of 16 to the corresponding carboxylic acid and its
esterification under standard conditions provided the methyl ester
derivative 17. Toward the desired target, the next requirement
was installation of the C-3 /C-3′ syn-diol moiety. In their studies
on amipurimycin, both Rauter and Garner research groups
have observed that, in the osmium tetroxide-catalyzed dihy-
droxylation of alkylidene pyrans structurally very similar to 17,
the bulky oxidizing reagent approaches the exocyclic olefin from
the less hindered ꢀ-face, resulting in stereoselective formation
(9) For similar examples, see: (a) Bhatt, R. K.; Chauhan, K.; Wheelan, P.;
Falck, J. R.; Murphy, R. C. J. Am. Chem. Soc. 1994, 116, 5050–5056. (b)
Reference 6b above.
4168 J. Org. Chem. Vol. 73, No. 11, 2008

Synthetic Studies on Amipurimycin
SCHEME 2
of the product diol.4b,5 Accordingly, employing the above
protocol, cis-dihydroxylation of the olefinic moiety of the
alkylidene pyran derivative 17, with a catalytic quantity of
osmium tetroxide, followed by acetylation (to facilitate chro-
matographic separation) of the crude diol resulted in the isolation
of the corresponding acetates 18 and 19, with the expected
product 18 being the major diastereoisomer (18:19 ) 82:18).
In accordance with the aforementioned literature precedence,
formation of the major diastereoisomer 18 is probably a result of
the sterically more favorable approach of the oxidant from the top
face of the olefin. Our subsequent attempts to further improve the
selectivity in the above dihydroxylation reaction, via employment
of the Sharpless asymmetric dihydroxylation (SAD) protocol,10
however, failed to produce any desired improvement. The failure
of SAD in the present substrate is most probably due to the steric
crowding around the trisubstituted exocyclic olefin 17 and its
unfavorable interactions with the bulky chiral catalyst.
With the completely functionalized carbohydrate amino acid core
of amipurimycin in hand, incorporation of the purine nucleobase
toward formation of the desired nucleoside fragment was initiated.
Thus, standard deprotection of the PMB protecting group of 18,
followed by acetylation of the resulting crude lactol yielded the
corresponding acetate 20 as an anomeric mixture of products
(Scheme 2). Employing the Vorbru¨ggen conditions,11 the nucleo-
side-forming reaction between the glycosyl donor 20 and the bis-
TMS-2-(N-acetylamino)purine 2112 was next undertaken. Disap-
pointingly, multiple attempts, involving different combinations of
Lewis acid activators/solvents and various reaction conditions
(Scheme 2), failed to provide the desired nucleoside, instead
resulting in either the recovery or extensive degradation (under
more rigorous conditions) of the starting material.
the above study was carried out on a structurally simplified
substrate lacking the branched chain at C-3 of the pyranosyl
carbohydrate core.
Being unable to achieve N-glycosidation of the pryranoside
20 under the traditional Vorbru¨ggen conditions, we decided to
explore alternative methods toward activation of the above
glycosyl donor and study its nucleoside-forming reaction.
Formation of activated glycosyl donors via the intermediacy of
highly reactive anomeric trichloroacetimidates (Schmidt’s trichlo-
roacetimidate protocol)13 is among the most commonly used
methods in contemporary O-glycosidic bond-forming reactions.
Surprisingly, the employment of this method in the synthesis
of nucleosides remains relatively infrequent.11 With the intent
to investigate the trichloroacetimidate protocol in our desired
nucleoside synthesis, the anomeric PMB-ether 18 was subjected
to standard CAN deprotection, followed by reaction of the
resulting crude lactol with trichloroacetonitrile, forming the
expected trichloroacetimidate derivative 23 (Scheme 3) in high
overall yield. This compound was, however, found to undergo
decomposition if left in the chromatographic column for an
extended period of time, or on prolonged storage. Therefore,
after rapid purification, compound 23 was used immediately
for the next reaction. Unfortunately, repeated attempts to couple
the trichloroacetimidate 23 with the bis-silylated 2-aminopurine
derivative 21 failed to result in the desired nucleoside formation.
Gratifyingly, reaction of 23 with bis-(TMS)thymine (30), in the
presence of TMSOTF as the Lewis acidic activator, finally led
to the highly stereoselective formation of the corresponding
thymine amipurimycin nucleoside 24, albeit in a modest yield
(Scheme 3). The characteristic coupling constant of the anomeric
proton (J1′,2′ ) 9.6 Hz)2 of a downstream product derived from
24 (compound 27, vide infra) helped confirm the H-1′/H-2′ trans-
relationship and the assigned C-1′ stereochemistry of the
nucleoside derivative 24. The anomeric stereoselectivity in the
above nucleoside-forming reaction can be rationalized by
invoking neighboring C-2′-acetoxy-assisted stabilization of the
oxonium ion intermediate and consequent blocking of the
R-face, directing the approach of the incoming nucleobase from
the opposite ꢀ-face.
Investigating the possibility of the above nucleoside formation
with a simpler pyrimidine nucleobase, reaction of 20 with
commercially available bis-TMS-thymine (22) was then at-
tempted. Once again the attempted reactions failed to form the
desired product. In their studies on amipurimycin, Czernecki
and co-workers did report the introduction of a 2-aminopurine
nucleoside to a model glycosyl amino acid donor.4d However,
(10) For recent reviews on Sharpless Asymmetric Dihydroxylation, see: (a)
Johnson, R. A.; Sharpless, K. B. Catalytic asymmetric dihydroxylation-discovery
and development. In Catalytic Asymmetric Synthesis, 2nd ed.; Ojima, I., Ed.;
Wiley-VCH: New York, 2000; pp 357-398. (b) Kolb, H. C.; Sharpless, K. B.
Asymmetric Dihydroxylation. In Transition Metals for Organic Synthesis, 2nd
ed.; Beller, M., Bolm, C., Eds.; Wiley-VCH: Weinheim, Germany, 2004; Vol.
2, pp 275-298. (c) Zaitsev, A. B.; Adolfsson, H. Synthesis2006, 172, 5–1756,
and references therein.
(11) Vorbru¨ggen, H.; Ruh-Pohlenz, C. Org. React. 2000, 55. 1–630, and
references therein.
(12) Garner, P.; Yoo, J. K.; Sarabu, R. Tetrahedron 1992, 21, 4259–4270.
Subsequently, peptidic attachment of the cispentacin side
chain was accomplished by hydrogenolysis of the N-Cbz
(13) (a) Schmidt, R. R.; Michel, J. J. Carbohydr. Chem. 1985, 4, 141–169.
(b) Schmidt, R. R. The anomeric O-alkylation and the trichloroacetimidate
methodsversatile strategies for glycoside bond formation. Front. Nat. Prod. Res.
1996, 1 (Modern Methods in Carbohydrate Synthesis), 20–54. (c) Schmidt, R. R.;
Jung, K.-H. Oligosaccharide Synthesis with Trichloroacetimidates. In PreparatiVe
Carbohydrate Chemistry; Hanessian, S., Ed.; Marcel Dekker Inc.: New York,
1997; pp 283-312, and references therein.
J. Org. Chem. Vol. 73, No. 11, 2008 4169

Stauffer and Datta
SCHEME 3
1
TABLE 1. Comparison of Selected H and 13C NMR Chemical
stereocenters in natural amipurimycin are probably in a (2′′R,3′′S)-
configuration, similar to the enantiopure (1R,2S)-cispentacin-
derived synthetic analogue 27.
Shifts of Amipurimycin and the Thymine Amipurimycin Analogue
27
thymine amipurimycin
In summary, utilizing a D-serine-derived enantiopure dihy-
dropyranone intermediate as a chiral platform, a robust and
efficient synthetic route to the complete glycosyl amino acid
core of amipurimycin has been developed. In terms of stereo-
controlled formation of new chiral centers and overall product
yield, the present method compares well with the previously
reported syntheses of the C-3′-branched carbohydrate structural
core of amipurimycin. Although our attempts to form the
2-amino purine containing nucleoside segment as present in the
natural product were unsuccessful, employing the trichloroace-
timidate glycosidation protocol, we could incorporate a pyri-
midine nucleobase to the above glycosyl core, thereby accom-
plishing the first total synthesis of a fully functionalized thymine
analogue of amipurimycin. Employing an alternative strategy
of introduction of the aminopurine nucleobase on a more simple,
early stage carbohydrate precursor (prior to C-3 branching), in
future studies, we plan to elaborate the present method toward
eventual completion of the total synthesis of amipurimycin, as
well as for SAR-targeted syntheses and biological evaluation
of strategically modified derivatives thereof.
δ (ppm)
amipurimycin (1) (in D2O)2
(27) (in D2O)
H-6′
C-6′
H-2′′
C-2′′
H-3′′
C-3′′
H-1′
C-1′
4.38(J)4Hz)
58.5
2.94
45.1
3.72
53.8
5.80(J)9Hz)
80.5
4.52(J)3.7Hz)
56.1
3.08
45.3
3.73
53.5
5.92(J)9.6Hz)
81.7
protecting group of 24 to unmask the free amine, which was
then coupled to (1R,2S)-N-trifluoroacetamido cispentacin deriva-
tive 254d to obtain the fully protected peptidyl nucleoside
structural core 26. Global removal of the protecting groups via
alkaline hydrolysis completed the total synthesis of the unique
thymine amipurimycin analogue 27. The structural assignment
and stereochemistry of the final product and its precursors were
confirmed on the basis of their spectral/analytical data and
literature precedence, wherever applicable.
Except for the nucleobase component, overlapping similarity
between the remaining structural features of natural amipuri-
mycin (1) and the thymine amipurimycin analogue 27 prompted
us to compare the NMR spectral data of the above compounds.
Comparison of the above data, related to the as yet undetermined
C-6′, C-2′′, and C-3′′ stereocenters of amipurimycin (1) and
the corresponding known stereocenters of the synthetic analogue
27, led to some interesting observations (Table 1).
As is evident, the proton and carbon chemical shift values
associated with the C-6′ R-amino acid stereocenter in amipu-
rimycin (1),2 as well as in the synthetic thymine analogue 27,
are very similar (Table 1). Additionally, the vicinal coupling
constant (J5′,6′) values for H-6′ in both the above compounds
are also strikingly close. The above data indicate that the
stereochemical configuration of the as yet unassigned C-6′
stereocenter in natural amipurimycin is probably the same (S)
as in the presently synthesized D-serine-derived analogue 27.
Along similar lines, the high conformity as seen between the
respective proton and carbon chemical shift values of the side
chain cispentacin stereocenters (2′′ and 3′′) in compounds 1 and
27 suggests that the hitherto undetermined C-2′′ and C-3′′
Experimental Section
(R)-Benzyl 4-((3aR,6S,7aR)-2,2-Dimethyl-4-oxotetrahydro-
3aH-[1,3]-dioxolo[4,5-c]pyran-6-yl)-2,2-dimethyloxazolidine-3-
carboxylate (8). To a stirring solution of the diol 76a (4.29 g, 11.8
mmol) in acetone (30 mL) and 2,2-dimethoxypropane (15 mL,
117.5 mmol) at room temperature was added BF3 ·OEt2 (0.1 mL,
0.7 mmol) dropwise. After being stirred at ambient temperature
for 3 h, the reaction was quenched with triethylamine (1 mL). The
solvent was removed under reduced pressure and the resulting oil
was purified by flash chromatography (hexanes/EtOAc ) 8:2 to
6:4) to afford the diacetonide derivative 8 as a viscous liquid (4.57
g, 96%): [R]D 88.4 (c 1.00, CHCl3); IR (NaCl) 1752, 1701 cm-1
;
1H NMR (400 MHz, CDCl3; mixture of rotamers) δ 1.28 and 1.35
(2 s, 3H), 1.39-1.63 (m, 9H), 1.93-2.14 (m, 2H), 3.94 and 3.97
(2 d, J ) 5.1 Hz, 1H), 4.03-4.22 (m, 2H), 4.52-4.68 (m, 3H),
5.09-5.23 (m, 2H), 7.36-7.42 (m, 5H); 13C NMR (125 MHz,
CHCl3; mixture of rotamers) δ 23.0, 23.9, 24.0, 24.5, 25.8, 26.0,
27.0, 27.8, 29.7, 31.8, 58.9, 59.7, 64.7, 65.3, 67.2, 67.7, 71.7, 71.8,
73.0, 74.2, 74.4, 94.5, 95.0, 110.8, 128.1, 128.5, 128.7, 135.8, 152.3,
153.9, 167.2, 167.4; HRMS calcd for C21H28NO7 m/z (M + H)+
406.1866, found 406.1876.
4170 J. Org. Chem. Vol. 73, No. 11, 2008

Synthetic Studies on Amipurimycin
(R)-Benzyl 4-((3aR,4R,6S,7aR)-4-(4-Methoxybenzyloxy)-2,2-
dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-6-yl)-2,2-di-
methyloxazolidine-3-carboxylate (9). Step 1: The lactone 8 (4.57
g, 11.28 mmol) was dissolved in anhydrous CH2Cl2 (60 mL) and
cooled to -78 °C. To this stirring solution was added DIBAL-H
(1.0 M in toluene, 13.5 mL, 13.5 mmol) dropwise. The reaction
was stirred at -78 °C for 1 h and then quenched by careful addition
of MeOH (1.0 mL). The reaction mixture was brought to rt and
diluted with EtOAc (80 mL), then saturated aq sodium potassium
tartrate solution (80 mL) was added. The resulting mixture was
stirred until two clear layers were seen. The layers were separated,
and the aqueous layer was extracted with EtOAc (3 × 25 mL).
The combined organic layers were dried (Na2SO4) and concentrated.
The crude lactol (4.72 g) thus obtained was carried on to the next
step without further purification.
and the residue was purified by flash chromatography (EtOAc/
hexanes ) 3:2) to afford the diol 11 as a white solid (0.368 g,
80%): mp 97-99 °C; [R]D -35.5 (c 0.20, CHCl3); IR (NaCl) 3442,
1
1717, 1701 cm-1; H NMR (500 MHz, CDCl3) δ 0.06 (s, 6H),
0.82 (s, 9H), 1.64 (t, J ) 12.1 Hz, 1H), 1.95 (d, J ) 14.5 Hz, 1H),
2.45 and 2.48 (2 s, 2H, exchangeable with D2O), 3.35 (br s, 1H),
3.59 (dd, J ) 3.4 and 9.9 Hz, 1H), 3.73 (br s, 4H), 3.87-3.95 (m,
2H), 4.11 (br s, 1H), 4.41 (d, J ) 11.2 Hz, 1H), 4.62 (d, J ) 7.9,
1H), 4.77 (d, J ) 11.2, 1H), 5.04 (s, 2H), 5.07 (d, J ) 9.4 Hz,
1H), 6.82 (d, J ) 8.6 Hz, 2H), 7.21 (d, J ) 8.5 Hz, 2H), 7.24-7.27
(m, 5H); 13C NMR (125 MHz, CDCl3) δ -5.2, -5.1, 18.5, 26.1,
34.1, 55.4, 55.5, 61.6, 67.0, 67.3, 69.2, 71.1, 71.9, 100.1, 114.2,
128.3, 128.4, 128.7, 129.6, 129.9, 136.7, 156.4, 159.7; HRMS calcd
for C29H43NO8SiNa m/z (M + Na)+ 584.2656, found 584.2655.
(2R,3R,4R,6S)-3-Acetoxy-4-hydroxy-2-(4-methoxybenzyloxy)-
6-((R)-8,8,9,9-tetramethyl-3-oxo-1-phenyl-2,7-dioxa-4-aza-8-si-
ladecan-5-yl)tetrahydro-2H-pyran (12). The diol 11 (1.35 g, 2.4
mmol) was dissolved in anhydrous CH2Cl2 (20 mL) and cooled to
0 °C. To this solution was added anhydrous pyridine (0.3 mL, 3.6
mmol) and DMAP (25 mg, catalytic), followed by Ac2O (0.25 mL,
2.6 mmol). The reaction mixture was stirred at 0 °C for 1 h and
then quenched with addition of ice-cooled water (5 mL). The two
layers were separated, and the aqueous layer was extracted with
CH2Cl2 (3 × 5 mL). The combined organic extracts were washed
with saturated aqueous NaHCO3 (1 × 10 mL) and brine (1 × 10
mL), dried with Na2SO4, and concentrated. The residue was purified
by flash chromatography (hexanes/EtOAc ) 3:2) to provide the
monoacetylated product 12 as a foamy solid (1.26 g, 88%): mp
108-110 °C; [R]D -52.3 (c 1.1, CHCl3); IR (NaCl) 3442, 1724
cm-1; 1H NMR (400 MHz, CDCl3) δ 0.09 and 0.10 (2 s, 6H), 0.92
(s, 9H), 1.82 (t, J ) 12.1 Hz, 1H), 2.03 (d, J ) 11.2 Hz, 1H), 2.10
(s, 4H, 1H exchangeable with D2O), 3.67 (dd, J ) 3.4 and 9.9 Hz,
1H), 3.79 (m, 1H), 3.82 (s, 3H), 4.02 (t, J ) 9.4 Hz, 2H), 4.28 (br
s, 1H), 4.53 (d, J ) 11.8 Hz, 1H), 4.82 (d, J ) 11.6 Hz, 2H), 4.87
(d, J ) 8.2 Hz, 1H), 5.12-5.16 (m, 3H), 6.88 (d, J ) 8.7, 2H),
7.24 (d, J ) 8.5 Hz, 2H), 7.33-7.39 (m, 5H); 13C NMR (125 MHz,
CDCl3) δ -5.4, -5.3, 18.3, 21.0, 25.9, 34.2, 55.1, 55.2, 61.4, 66.6,
66.9, 68.8, 70.4, 73.0, 97.6, 113.8, 128.1, 128.2, 128.5, 129.1, 129.7,
136.5, 156.1, 159.3, 169.4; HRMS calcd for C31H45NO9SiNa m/z
(M + Na)+ 626.2761, found 626.2759.
Step 2: The lactol (4.7 g, 11.6 mmol) as obtained above was
dissolved in anhydrous CH2Cl2 (80 mL). To this stirring solution
at room temperature was added tetrabutylammonium iodide (5.14
g, 13.9 mmol) and freshly prepared Ag2O (4.0 g, 17.4 mmol),
followed by p-methoxybenzyl bromide (1.8 mL, 12.8 mmol). The
resulting brown mixture was stirred for 2.5 h, then the solids were
filtered off and washed with CH2Cl2 (100 mL). The combined
filtrate was concentrated, redissolved in CH2Cl2 (40 mL), and
washed with 10% sodium thiosulfate (1 × 10 mL), then the aqueous
layer was back extracted with CH2Cl2 (1 × 10 mL). The combined
organic layers were dried (Na2SO4) and concentrated under vacuum,
then the residue was purified by flash chromatography (hexanes/
EtOAc ) 8:2) to yield the PMB-glycoside 9 as a colorless oil (4.94
g, 83% over two steps): [R]D -18.2 (c 0.75, CHCl3); IR (NaCl)
1
1703 cm-1; H NMR (400 MHz, CDCl3; mixture of rotamers) δ
1.34-1.40 (4 s, 6H), 1.60 and 1.67 (2 s, 6H), 1.76-1.90 (m, 1H),
2.03 (br s, 1H), 3.81 (s, 3H), 3.87-4.07 (m, 3H), 4.09-4.19 (m,
2H), 4.34 and 4.47 (2 br s, 1H), 4.53-4.60 (m, 2H), 4.79 (t, J )
12.1 Hz, 1H), 5.13-5.18 (m, 2H), 6.88 (d, J ) 8.2 Hz, 2H),
7.32-7.40 (m, 2H), 7.38 (br s, 5H); 13C NMR (125 MHz, CDCl3;
mixture of rotamers) δ 23.2, 24.6, 25.4, 25.5, 26.7, 27.4, 27.5, 27.7,
29.6, 29.7, 29.9, 55.3, 60.1, 61.0, 64.8, 65.2, 66.9, 67.4, 69.4, 69.6,
69.9, 71.8, 72.0, 74.7, 74.8, 94.3, 94.7, 99.6, 109.0, 113.8, 128.1,
128.2, 128.3, 128.6, 129.4, 129.5, 136.0, 152.5, 153.8, 159.3; HRMS
calcd for C29H38NO8 m/z (M + H)+ 528.2598, found 528.2568.
Benzyl (R)-1-((2S,4R,5R,6R)-4,5-Dihydroxy-6-(4-methoxyben-
zyloxy)tetrahydro-2H-pyran-2-yl)-2-hydroxyethylcarbamate
(10). The diacetonide 9 (1.4 g, 2.7 mmol) was dissolved in acetic
acid/water (30 mL, 3:1) and heated at 40 °C for 12 h. Excess solvent
was removed under reduced pressure, and the residue was purified
by flash chromatography (CHCl3/MeOH ) 97:3 to 95:5) to afford
the triol 10 as a white solid (1.07 g, 83%): mp 117-119 °C; [R]D
(2R,3S,6S)-3-Acetoxy-2-(4-methoxybenzyloxy)-6-((R)-8,8,9,9-
tetramethyl-3-oxo-1-phenyl-2,7-dioxa-4-aza-8-siladecan-5-yl)tet-
rahydro-2H-pyran-4-one (13). To an ice-cooled anhydrous CH2Cl2
solution (30 mL) of the alcohol 12 (1.2 g, 2.0 mmol) was added
Dess-Martin periodinane (15% in CH2Cl2, 8 mL, 2.8 mmol)
dropwise. The resulting solution was stirred at 0 °C for 0.5 h, the
ice bath was removed, and the reaction was allowed to attain rt.
After being stirred for another 2.5 h, the reaction was quenched by
addition of saturated aq NaHCO3 (20 mL) and solid sodium
thiosulfate (3.0 g) followed by its dilution with EtOAc (50 mL).
After allowing the mixture to stir until clear separation of the
organic layer, the two layers were separated, and the aqueous layer
was extracted with EtOAc (3 × 10 mL). The combined organic
extracts were washed sequentially with saturated aqueous NaHCO3
(1 × 20 mL) and brine (1 × 20 mL), dried (Na2SO4), and
concentrated under vacuum. The residue was purified by flash
chromatography to afford the ketone 13 as a viscous oil (1.09 g,
-44.4 (c 0.90, MeOH); IR (Teflon film) 3506, 3325, 1683 cm-1
;
1H NMR (400 MHz, CD3OD) δ 1.61 (t, J ) 13.9 Hz, 1H), 1.87 (d,
J ) 14.1 Hz, 1H), 3.66-3.74 (m, 4H), 3.76 (s, 3H), 3.82-3.93
(m, 1H), 4.08 (br s, 1H), 4.57 (d, J ) 11.4 Hz, 1H), 4.67 (d, J )
8.0 Hz, 1H), 4.76 (d, J ) 11.0 Hz, 1H), 5.07 and 5.12 (2 d, J )
6.3 Hz, 2H), 6.87 (d, J ) 8.0 Hz, 2H), 7.28-7.38 (m, 7H); 13C
NMR (125 MHz, CD3OD) δ 36.1, 55.8, 57.8, 62.4, 67.7, 69.2, 70.9,
71.6, 72.8, 101.2, 114.8, 129.0, 129.1, 129.6, 131.0, 131.4, 138.5,
158.9, 161.0; HRMS calcd for C23H30NO8 m/z (M + H)+ 448.1971,
found 448.1977.
Benzyl (R)-2-(tert-Butyldimethylsilyloxy)-1-((2S,4R,5R,6R)-
4,5-dihydroxy-6-(4-methoxybenzyloxy)tetrahydro-2H-pyran-2-
yl)ethylcarbamate (11). To a stirred solution of the triol 10 (0.37
g, 0.82 mmol) in anhydrous DMF (6 mL), DMAP (25 mg,
catalytic), and imidazole (0.12 g, 1.8 mmol) was added TBDMSCl
(0.15 g, 0.98 mmol). The resulting solution was heated at 70 °C
for 12 h. After cooling to room temperature, the reaction was
quenched by addition of H2O (10 mL), followed by dilution of the
mixture by ether (20 mL), and stirred for 5 min. The two layers
were separated and the aqueous layer was extracted with ether (3
× 10 mL). The combined organic extracts were washed with brine
(1 × 20 mL), dried with Na2SO4, and concentrated under vacuum,
91%): [R]D -61.6 (c 1.28, CHCl3); IR (NaCl) 1753, 1731 cm-1
;
1H NMR (400 MHz, CDCl3) δ 0.07 (s, 6H), 0.89 (s, 9H), 2.14 (s,
3H), 2.61-2.76 (m, 2H), 3.66-3.70 (m, 2H), 3.80 (s, 3H),
3.91-3.98 (m, 1H), 4.02 (d, J ) 9.9 Hz, 1H), 4.58 (d, J ) 11.6
Hz, 1H), 4.67 (d, J ) 8.2 Hz, 1H), 4.84 (d, J ) 11.6, 1H),
5.11-5.14 (m, 4H), 6.88 (d, J ) 8.6 Hz, 2H), 7.24 (d, J ) 8.6 Hz,
2H), 7.35 (br s, 5H); 13C NMR (125 MHz, CDCl3) δ -5.5, -5.4,
18.2, 20.5, 25.8, 44.0, 55.2, 55.3, 61.2, 67.2, 70.3, 71.0, 77.9, 100.6,
113.9, 128.2, 128.4, 128.6, 128.7, 129.4, 136.1, 156.0, 159.5, 169.4,
199.1; HRMS calcd for C31H43NO9SiNa m/z (M + Na)+ 624.2605,
found 624.2587.
J. Org. Chem. Vol. 73, No. 11, 2008 4171

Stauffer and Datta
Benzyl (R)-2-(tert-Butyldimethylsilyloxy)-1-((2S,5R,6R,E)-5-
hydroxy-4-((E)-methoxycarbonylidene)-6-(4-methoxybenzyloxy)-
tetrahydro-2H-pyran-2-yl)ethylcarbamate (14). To a stirred
solution of the ketone 13 (0.186 g, 0.31 mmol) in anhydrous
benzene (6 mL) was added (carbomethoxymethylene)triphe-
nylphosphorane (0.22 g, 0.62 mmol). The resulting solution was
refluxed at 80 °C for 12 h and then the solvent was removed under
vacuum. The resulting residue was purified by flash chromatography
(hexanes/EtOAc ) 4:1) to yield the E-alkylidene ester 14 as a
viscous oil (0.197 g, 95%): [R]D -56.3 (c 0.50, CHCl3); IR (NaCl)
solution (10 mL). After the mixture was diluted with EtOAc (25
mL), the two layers were separated, and the aqueous layer was
extracted with EtOAc (3 × 10 mL). The combined organic extracts
were washed with brine (1 × 25 mL), dried with Na2SO4, and
concentrated under vacuum. The crude residue was purified by flash
chromatography (EtOAc/hexanes ) 3:2) to afford the alcohol 16
as a foamy solid (0.786 g, 96%): mp ) 94-96 °C; [R]D -44.40 (c
1
1.0, CHCl3); IR (NaCl) 3350, 1736 cm-1; H NMR (400 MHz,
CDCl3) δ 2.06 (s, 3H), 2.11 (s, 1H), 2.15 (s, 3H), 2.40 (br s, 1H,
exchangeable with D2O), 2.80 (d, J ) 12.4 Hz, 1H), 3.55-3.65
(m, 1H), 3.69-3.77 (m, 1H), 3.79 (s, 4H), 3.98 (d, J ) 10.7 Hz,
1H), 4.33 (d, J ) 7.8 Hz, 1H), 4.49-4.74 (m, 4H), 5.15 (s, 2H),
5.24 (d, J ) 7.5 Hz, 1H), 5.48-5.54 (m, 2H), 6.87 (d, J ) 8.4 Hz,
2H), 7.22 (d, J ) 8.4 Hz, 2H), 7.34-7.40 (m, 5H); 13C NMR (100.6
MHz, CDCl3) δ 20.9, 21.0, 31.1, 54.6, 55.3, 59.9, 61.4, 67.0, 70.7,
72.6, 75.6, 101.6, 113.9, 117.4, 128.2, 128.3, 128.6, 129.2, 129.4,
135.8, 136.3, 156.3, 159.4, 169.5, 170.9; HRMS calcd for
C29H35NO10Na m/z (M + Na)+ 580.2158, found 580.2050.
(S)-Methyl 2-(Benzyloxycarbonylamino)-2-((2S,5R,6R,E)-5-
(acetoxy)-4-(2-(acetoxyethylidene)-6-(4-methoxybenzyloxy)tet-
rahydro-2H-pyran-2-yl)ethanoate (17). To an ice-cooled anhy-
drous CH2Cl2 solution (10 mL) of the alcohol 16 (0.73 g, 1.3 mmol)
was added Dess-Martin periodinane (15% in CH2Cl2, 5.0 mL, 1.8
mmol) dropwise. The reaction was stirred at 0 °C for 0.5 h, the
ice-bath was removed, and the reaction was allowed to attain rt.
After being stirred for another 2.5 h, the reaction was diluted with
EtOAc (30 mL) and quenched by the addition of saturated aq
NaHCO3 (10 mL) solution and solid sodium thiosulfate (2.0 g).
After the mixture was stirred until the organic layer was mostly
clear, the two layers were separated, and the aqueous layer was
extracted with EtOAc (3 × 10 mL). The combined organic extracts
were washed sequentially with saturated aqueous NaHCO3 (1 ×
20 mL) and brine (1 × 20 mL), dried (Na2SO4), and concentrated
under vacuum. The crude aldehyde thus obtained (∼0.7 g) was
carried on to the next reaction without further purification.
Step 2: The crude aldehyde (0.7 g, 1.26 mmol) was dissolved
in tert-butyl alcohol (50 mL) and 2-methyl-2-butene (2 M in THF,
33 mL, 66 mmol). To this stirring mixture was slowly added a
solution of sodium chlorite (1.23 g, 13.6 mmol) and sodium
dihydrogen phosphate (1.26 g, 10.5 mmol) dissolved in water (20
mL). The biphasic reaction was stirred vigorously at room tem-
perature for 30 min. The two layers were separated, and the aqueous
layer was extracted with EtOAc (3 × 10 mL). The combined
organic extracts were dried (Na2SO4) and concentrated to afford
the crude acid as colorless oil (∼0.8 g), which was taken on to the
next step without any further purification.
1
1751, 1722 cm-1; H NMR (400 MHz, CDCl3) δ 0.09 (s, 6H),
0.91 (s, 9H), 2.14 (s, 3H), 2.17-2.24 (m, 1H), 3.64 (br t, J ) 9.5
Hz, 1H), 3.69 (br s, 4H), 3.81 (s, 3H), 3.89-3.96 (m, 1H), 4.00 (d,
10.0 Hz, 1H), 4.14 (d, J ) 14.2, 1H), 4.40 (d, J ) 7.7 Hz, 1H),
4.55 (d, J ) 11.8 Hz, 1H), 4.81 (d, J ) 11.8 Hz, 1H), 5.15 and
5.21 (2 d, J ) 12.2 Hz, 3H), 5.33 (d, J ) 7.5 Hz, 1H), 5.77 (s,
1H), 6.88 (d, J ) 8.8 Hz, 2H), 7.23 (d, J ) 8.6 Hz, 2H), 7.34-7.39
(m, 5H); 13C NMR (125 MHz, CDCl3) δ -5.4, -5.3, 18.3, 20.8,
25.9, 31.7, 51.3, 55.3, 61.4, 66.9, 70.3, 72.9, 73.0, 77.3, 101.4,
113.2, 113.8, 128.0, 128.1, 128.5, 129.2, 136.5, 151.4, 156.1, 159.3,
166.1, 169.2; HRMS calcd for C34H48NO10Si m/z (M + H)+
658.3047, found 658.3051.
Benzyl (R)-2-(tert-Butyldimethylsilyloxy)-1-((2S,5R,6R,E)-5-
acetoxy-4-(2-acetoxyethylidene)-6-(4-methoxybenzyloxy)tetrahy-
dro-2H-pyran-2-yl)ethylcarbamate (15). Step 1: The alkylidene
ester 14 (1.47 g, 2.2 mmol) was dissolved in anhydrous CH2Cl2
(15 mL) and cooled to -40 °C. To this stirring solution was added
DIBAL-H (1.0 M in toluene, 7.0 mL, 7.0 mmol) dropwise. The
reaction was stirred at -40 °C for 1 h and upon completion of the
reaction (TLC monitoring often necessitated an additional 1.0 equiv
of DIBAL-H to be added) was quenched by careful addition of
MeOH (2.0 mL). The reaction mixture was brought to rt and diluted
with EtOAc (50 mL), then saturated aqueous sodium potassium
tartrate (25 mL) was added. The resulting mixture was stirred until
two clear layers were seen. The two layers were separated, and the
aqueous layer was extracted with EtOAc (3 × 30 mL). The
combined extracts were washed with brine, dried (Na2SO4), and
concentrated under vacuum. The residue thus obtained (1.12 g) was
directly used for the next reaction without any further purification.
Step 2: The crude diol (1.12 g, 1.87 mmol) as obtained from
above was dissolved in anhydrous CH2Cl2 (15 mL) and treated with
pyridine (0.8 mL, 9.3 mmol), DMAP (20 mg, catalytic), and Ac2O
(0.9 mL, 9.3 mmol). The resulting mixture was stirred at rt for 2 h
followed by quenching of the reaction by addition of ice-cooled
water (10 mL). The two layers were separated and the aqueous
layer was extracted with CH2Cl2 (3 × 10 mL). The combined
organic extracts were washed with saturated aqueous NaHCO3 (1
× 5 mL) and brine (1 × 5 mL), dried with Na2SO4, and
concentrated under vacuum. The crude product obtained was
purified by flash chromatography (hexanes/EtOAc ) 7:3) affording
the diacetate 15 as a low-melting solid (1.16 g, 79% over 2 steps):
[R]D -47.4 (c 0.50, CHCl3); IR (NaCl) 1740 cm-1; 1H NMR (500
MHz, CDCl3) δ 0.01 (s, 6H), 0.83 (s, 9H), 1.97 (s, 3H), 2.04 (br
s, 4H), 2.74 (d, J ) 13.0 Hz, 1H), 3.37 (t, J ) 9.4 Hz, 1H), 3.6
(dd, J ) 3.3 and 10.0 Hz, 1H), 3.73 (s, 3H), 3.77 (br s, 1H), 3.92
(d, J ) 9.9 Hz, 1H), 4.28 (d, J ) 7.7 Hz, 1H), 4.39-4.46 (m, 2H),
4.55-4.59 (m, 1H), 4.71 (d, J ) 11.9 Hz, 1H), 5.04-5.17 (m,
4H), 5.39 (t, J ) 7.0 Hz, 1H), 6.8 (d, J ) 8.6 Hz, 2H), 7.14 (d, J
) 8.5, 2H), 7.25-7.31 (m, 5H); 13C NMR (125 MHz, CDCl3) δ
-5.3, -5.2, 18.5, 21.1, 21.2, 26.1, 31.3, 55.5, 56.0, 60.2, 61.5,
67.1, 70.4, 73.0, 73.3, 101.6, 114.0, 117.3, 128.3, 128.4, 128.8,
129.3, 129.6, 136.5, 136.7, 156.4, 159.5, 169.6, 170.0; HRMS calcd
for C35H50NO10Si m/z (M + H)+ 672.3204, found 672.3201.
Benzyl (R)-2-Hydroxy-1-((2S,5R,6R,E)-5-acetoxy-4-(2-acetoxy-
ethylidene)-6-(4-methoxybenzyloxy)tetrahydro-2H-pyran-2-yl)-
ethylcarbamate (16). To a stirred solution of 15 (0.99 g, 1.48
mmol) in anhydrous THF (10 mL) at 0 °C was added TBAF (1 M
in THF, 2.0 mL, 2 mmol). The reaction was stirred for 3 h at 0 °C,
and then quenched with saturated aqueous ammonium chloride
Step 3: [Caution: Diazomethane is an explosive and a highly
toxic gas. Explosions may occur if the substance is dry and
undiluted. All operations involving diazomethane should be carried
out in an efficient fume hood following appropriate precaution.]
To an ice-cooled biphasic solution of KOH (2 g in 8 mL of water)
and ether (20 mL) was added N-methyl-N′-nitro-N-nitrosoguanidine
(MNNG, 1 g) in one lot. The organic layer turned bright yellow.
The ethereal layer was decanted into an ice-cooled Erlenmeyer flask
containing KOH pellets. The aqueous layer was washed with ether
(3 × 15 mL), and the ethereal layers were combined. The
diazomethane (CH2N2) thus prepared was added to a stirred solution
of the crude acid (0.8 g in 10 mL of ether) as obtained from step
2 above, and stirring was continued at room temperature for 30
min. Excess CH2N2 was removed by bubbling nitrogen into the
reaction mixture for 15 min, followed by removal of solvent under
vacuum to yield the crude product. Purification by flash chroma-
tography (hexanes/EtOAc ) 3:2) provided the methyl ester 17 as
an oil (0.7 g, 85% over 3 steps): [R]D -29.62 (c 1.0, CHCl3); IR
1
(NaCl) 3367, 1736 cm-1; H NMR (500 MHz, CDCl3) δ 2.04 (s,
3H), 2.12 (s, 3H), 2.32 (t, J ) 13.0 Hz, 1H), 2.80 (d, J ) 12.5 Hz,
1H), 3.63 (br d, J ) 11.7 Hz, 1H), 3.78 (s, 3H), 3.79 (s, 3H), 4.31
(d, J ) 7.6 Hz, 1H), 4.48-4.56 (m, 3H), 4.60-4.66 (m, 1H), 4.70
(d, J ) 15.0 Hz, 1H), 5.14 (s, 2H), 5.19 (d, J ) 7.4 Hz, 1H), 5.49
4172 J. Org. Chem. Vol. 73, No. 11, 2008

Synthetic Studies on Amipurimycin
(t, J ) 6.9 Hz, 1H), 5.62 (d, J ) 8.5 Hz, 1H), 6.85 (d, J ) 8.4 Hz,
2H), 7.19 (d, J ) 8.6 Hz, 2H), 7.32-7.38 (m, 5H); 13C NMR (125
MHz, CDCl3) δ 20.8, 21.0, 30.7, 52.7, 55.2, 56.9, 59.8, 67.3, 69.9,
72.4, 75.1, 100.8, 113.8, 117.9, 128.2, 128.3, 128.6, 129.1, 129.4,
135.4, 136.0, 155.7, 159.3, 169.5, 169.7, 170.8; HRMS calcd for
C30H35NO11Na m/z (M + Na)+ 608.2108, found 608.2096.
Dihydroxylation and Acetylation of 17 To Form the Triac-
etates 18 and 19. Step 1: The allylic acetate 17 (0.66 g, 1.12 mmol)
was dissolved in acetone/water (10 mL, 4:1) and cooled to 0 °C.
To this stirring solution was added NMO·H2O (0.38 g, 2.8 mmol),
followed by OsO4 (5% in toluene, 0.4 mL, 0.08 mmol). The
resulting solution was stirred at 0 °C for 6 h. After completion of
the reaction (TLC monitoring), 10% aqueous Na2SO3 (5 mL) was
added to the reaction mixture along with EtOAc (15 mL). The
organic layer was separated, and the aqueous layer was extracted
with EtOAc (3 × 5 mL). The combined organic extracts were dried
with NasSO4 and concentrated under vacuum. The crude diaster-
eomeric mixture of product diols (0.63 g) was taken on to the next
reaction without any further purification.
acetonitrile/water (5 mL, 9:1) was added ceric ammonium nitrate
(0.32 g, 0.59 mmol). The reaction was stirred at rt for 1.5 h and
then poured into a separatory funnel and diluted with EtOAc (10
mL). The organic layer was washed sequentially with water (5 mL),
saturated aqueous NaHSO3 (5 mL), and saturated aqueous NaHCO3
(5 mL). The organic layer was dried with Na2SO4 and concentrated.
The crude lactol (0.1 g) thus obtained was taken on to the next
reaction without further purification.
Step 2: The above lactol (0.104 g, 0.19 mmol) was dissolved in
anhydrous CH2Cl2 (2 mL), and to this solution was added anhydrous
pyridine (0.04 mL, 0.5 mmol), DMAP (10 mg, catalytic), followed
by Ac2O (0.03 mL, 0.32 mmol). The reaction was stirred at rt for
2 h and then quenched with addition of ice-cooled water (5 mL).
The two layers were separated, and the aqueous layer was extracted
with CH2Cl2 (3 × 5 mL). The combined organic extracts were
washed with saturated aqueous NaHCO3 (1 × 10 mL) and brine
(1 × 10 mL), dried with Na2SO4, and concentrated under vacuum.
The residue was purified by flash chromatography (hexanes/EtOAc
) 2:3) to yield the anomeric mixture of the triacetate 20 as a
1
semisolid (0.1 g, 95% over 2 steps): H NMR (400 MHz, CDCl3;
Step 2: The mixture of diols (0.64 g, 1.03 mmol) as obtained
from the above reaction was dissolved in anhydrous CH2Cl2 (10
mL), and to this solution was added sequentially anhydrous pyridine
(0.14 mL, 1.7 mmol), DMAP (25 mg, catalytic), and Ac2O (0.15
mL, 1.5 mmol). The reaction was stirred at rt for 2 h and then
quenched with ice-cooled water (5 mL). The two layers were
separated, and the aqueous layer was extracted with CH2Cl2 (3 ×
5 mL). The combined organic extracts were washed with saturated
aqueous NaHCO3 (1 × 10 mL) and brine (1 × 10 mL), dried with
Na2SO4, and concentrated under vacuum. The mixture of diaster-
eomeric products was separated by flash chromatography (CHCl3/
EtOAc ) 9:1 to 4:1) to afford the corresponding triacetates 18
(major) and 19 (minor) (ca. 4:1 ratio) in 94% overall yield.
(S)-1-((2R,3R,4R,6S)-6-((S)-1-(Benzyloxycarbonylamino)-2-
methoxy-2-oxoethyl)-3-(ethanoyloxy)-4-hydroxy-2-(4-methoxy-
benzyloxy)-tetrahydro-2H-pyran-4-yl)ethane-1,2-diyl dietha-
noate (18). Major isomer: foamy solid (0.57 g, 77%), mp ) 44-46
mixture of anomers) δ 1.93-2.22 (m, 14H), 3.75 and 3.78 (2 s,
3H), 3.91-4.27 (m, 2H), 4.38 (br d, J ) 9.3 Hz, 1H), 4.85-5.30
(m, 6H), 5.61-5.69 (m, 1H), 5.95 and 6.44 (2 s, 1H), 7.33-7.42
(m, 5H); 13C NMR (125.7 MHz, CDCl3; mixture of anomers) δ
20.5, 20.7, 20.8, 20.9, 21.0, 22.4, 26.2, 31.2, 31.6, 52.6, 52.7, 56.6,
63.2, 63.5, 63.8, 67.3, 67.7, 69.1, 69.6, 69.9, 70.1, 70.8, 71.8, 72.7,
79.4, 80.2, 89.2, 90.0, 128,1, 128.2, 128.3, 128.6, 128.8, 128.9,
136.0, 155.8, 168.7, 168.9, 169.0, 169.2, 169.3, 169.5, 169.7, 169.8,
170.7, 170.9; HRMS calcd for C26H33NO14Na m/z (M + Na)+
606.1799, found 606.1755.
(S)-1-((3R,4R,6S)-6-((S)-1-(Benzyloxycarbonylamino)-2-meth-
oxy-2-oxoethyl)-3-(ethanoyloxy)-4-hydroxy-2-(2,2,2-trichloro-1-
iminoethoxy)tetrahydro-2H-pyran-4-yl)ethane-1,2-diyl Dietha-
noate (23). Step 1: To a stirring, room temperature solution of the
PMB-glycoside 18 (0.28 g, 0.42 mmol) in acetonitrile/water (10
mL, 9:1) was added ceric ammonium nitrate (0.65 g, 1.19 mmol).
After being stirred for 1.5 h the reaction mixture was poured into
a separatory funnel and diluted with EtOAc (20 mL). The organic
layer was washed sequentially with water (10 mL), saturated
aqueous NaHSO3 (5 mL), and saturated aqueous NaHCO3 (5 mL).
The organic layer was dried with Na2SO4 and concentrated. The
crude lactol (0.22 g) was taken on to the next reaction without
further purification.
1
°C; [R]D -45.2 (c 1.95, CHCl3); IR (NaCl) 3350, 1744 cm-1; H
NMR (400 MHz, CDCl3) δ 2.03, 2.04 and 2.07 (3 s, 9H), 2.01-2.22
(m, 2H), 3.59 (br s, 1H, exchangeable with D2O), 3.81 and 3.82 (2
s, 6H), 4.08 and 4.11 (dd, J ) 8.3 and 12.2 Hz, 1H), 4.18-4.25
(m, 1H), 4.45 (d, J ) 11.4 Hz, 1H), 4.53 (dd, J ) 2.6 and 12.2 Hz,
1H), 4.65-4.70 (m, 1H), 4.74 (d, J ) 11.4 Hz, 1H), 4.84 (d, J )
2.8 Hz, 1H), 4.90 (d, J ) 2.7 Hz, 1H), 5.15 (br s, 2H), 5.23 (dd,
J ) 1.8 and 8.2 Hz, 1H), 5.62 (d, J ) 8.7 Hz, 1H), 6.87 (d, J )
8.6 Hz, 2H), 7.23 (d, J ) 8.6 Hz, 2H), 7.28-7.47 (m, 5H); 13C
NMR (100.6 MHz, CDCl3) δ 20.8, 21.0, 33.5, 52.7, 55.3, 57.2,
62.6, 67.4, 70.2, 70.4, 71.1, 71.7, 72.3, 98.2, 113.9, 128.3, 128.4,
128.6, 129.6, 135.9, 156.0, 159.5, 169.7, 170.0, 170.3, 171.0; HRMS
calcd for C32H39NO14Na m/z (M + Na)+ 684.2268, found 684.2236.
(R)-1-((2R,3R,4S,6S)-6-((S)-1-(Benzyloxycarbonylamino)-2-
methoxy-2-oxoethyl)-3-(ethanoyloxy)-4-hydroxy-2-(4-methoxy-
benzyloxy)tetrahydro-2H-pyran-4-yl)ethane-1,2-diyl Dietha-
noate (19). Minor isomer: foamy solid (0.126 g, 17%), mp 56-58
Step 2: The above crude lactol (0.22 g, 0.41 mmol) was dissolved
in anhydrous CH2Cl2 (8 mL), and to this solution was added
anhydrous K2CO3 (0.2 g, 1.45 mmol), followed by dropwise
addition of trichloroacetonitrile (0.4 mL, 4.0 mmol). The reaction
was stirred at room temperature for 12 h and then quenched with
water (5 mL). The two layers were separated and the aqueous layer
was extracted with EtOAc (3 × 10 mL). The combined organic
layers were washed with brine (1 × 10 mL), dried (Na2SO4), and
concentrated. The residue was quickly purified by flash chroma-
tography (EtOAc/hexanes ) 1:1) to afford the trichloroacetimidate
23 as a viscous liquid (0.256 g, 89% over 2 steps). The compound
was found to be unstable and was used immediately after purifica-
1
°C; [R]D -14.79 (c 1.9, CHCl3); IR (NaCl) 3354, 1744 cm-1; H
NMR (400 MHz, CDCl3) δ 1.89-1.99 (m, 2H), 2.05 (s, 3H), 2.12
(s, 6H), 2.50 (s, 1H, exchangeable with D2O), 3.80 (s, 6H),
4.02-4.13 (m, 2H), 4.36 (d, J ) 9.7 Hz, 1H), 4.46-4.52 (m, 2H),
4.70-4.73 (m, 2H), 4.90 (d, J ) 7.6 Hz, 1H), 5.09 (dd, J ) 2.8
and 7.5 Hz, 1H), 5.18 (s, 2H), 5.58 (d, J ) 8.8 Hz, 1H), 6.86 (d,
J ) 8.3 Hz, 2H), 7.18 (d, J ) 8.4 Hz, 2H), 7.39 (br s, 5H); 13C
NMR (100.6 MHz, CDCl3) δ 20.7, 20.9, 21.0, 33.6, 52.7, 55.3,
56.9, 61.8, 67.3, 70.5, 71.4, 71.6, 73.3, 73.9, 98.3, 113.8, 128.3,
128.4, 128.6, 129.2, 129.3, 136.0, 155.8, 159.3, 169.5, 169.7, 170.6,
171.1; HRMS calcd for C32H40NO14 m/z (M + H)+ 662.2449, found
662.2459.
tion: IR (NaCl) 3336, 1745, 1677 cm-1 1H NMR (400 MHz,
;
CDCl3; mixture of anomers) δ 2.05, 2.06, 2.07 and 2.10 (4 s, 9H),
2.14-2.30 (m, 2H), 3.73 and 3.76 (2 s, 3H), 4.05-4.43 (m, 4H),
4.92-5.17 (m, 4H), 5.41 (s, 1H), 5.61-5.70 (m, 1H), 6.08 and
6.58 (2 s, 1H), 7.37 (br s, 5H), 8.58 and 8.68 (2 s, 1H); 13C NMR
(125.7 MHz, CDCl3; mixture of anomers) δ 20.7, 20.8, 29.7, 52.8,
55.3, 62.1, 62.2, 65.7, 67.8, 72.1, 72.9, 90.8, 92.2, 95.1, 128.3,
128.4, 128.5, 128.6, 135.6, 156.4, 160.0, 169.5, 170.4, 170.7; HRMS
calcd for C26H31Cl3N2O13Na m/z (M + Na)+ 707.0789, found
707.0741.
(1R)-1-((3R,4S,6S)-6-((S)-1-(Benzyloxycarbonylamino)-2-meth-
oxy-2-oxoethyl)-2,3-bis(ethanoyloxy)-4-hydroxytetrahydro-2H-
pyran-4-yl)ethane-1,2-diyl Diethanoate (20). Step 1: To a stirring
solution of the PMB-glycoside 18 (0.125 g, 0.19 mmol) in
(S)-1-((2R,3R,4R,6S)-6-((S)-1-(Benzyloxycarbonylamino)-2-
methoxy-2-oxoethyl)-3-(ethanoyloxy)-4-hydroxy-2-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydro-2H-pyran-4-
yl)ethane-1,2-diyl diethanoate (24). To a solution of trichloroace-
J. Org. Chem. Vol. 73, No. 11, 2008 4173

Stauffer and Datta
timidate 24 (0.163 g, 0.24 mmol) in anhydrous 1,2-dichloroethane
(8 mL) was added commercially available bis(trimethylsilyl)thymine
(22) (0.32 g, 1.2 mmol), followed by freshly distilled TMSOTf
(0.17 mL, 0.96 mmol). The reaction was stirred at rt for 1 h, and
then saturated aqueous NaHCO3 (5 mL) solution was added to
quench the reaction. The precipitated excess nucleobase was filtered
off and the white solid was washed with CHCl3 (3 × 5 mL). The
combined filtrate was transferred to a separating funnel and the
two layers were separated. The aqueous layer was extracted with
CHCl3 (3 × 5 mL) and combined organic extracts were dried with
Na2SO4 and concentrated under vacuum. The residue was purified
by flash chromatography (EtOAc/hexanes ) 7:3 to 9:1) to afford
the thymine nucleoside 24 as a white solid (0.051 g, 33%): mp
107-109 °C; [R]D -6.2 (c 1.35, CHCl3); IR (NaCl) 3296, 1744,
1697 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.88 (s, 3H), 2.05, 2.06,
and 2.10 (3 s, 9H), 2.16-2.29 (m, 2H), 3.80 (s, 3H), 4.07 (s, 1H,
exchangeable with D2O), 4.19 (dd, J ) 7.7 and 11.9 Hz, 1H), 4.25
(br s, 1H), 4.55 (dd, J ) 2.7 and 12.0 Hz, 1H), 4.61 (d, J ) 6.6
Hz, 1H), 5.05 (d, J ) 5.2 Hz, 1H), 5.14 (s, 2H), 5.41 (d, J ) 4.7
Hz, 1H), 5.80 (d, J ) 4.4 Hz, 1H), 6.02 (br s, 1H), 7.27 (s, 1H),
7.36 (br s, 5H), 8.92 (s, 1H); 13C NMR (100.6 MHz, CDCl3) δ
12.6, 20.7, 20.8, 20.9, 33.2, 53.0, 57.2, 62.6, 67.4, 72.1, 72.7, 72.8,
77.3, 111.4, 128.2, 128.4, 128.6, 135.9, 136.1, 150.1, 155.9, 163.4,
169.7, 169.8, 169.9, 170.9; HRMS calcd for C29H36N3O14 m/z (M
+ H)+ 650.2219, found 650.2179.
(S)-1-((2R,3R,4R,6S)-3-(Ethanoyloxy)-4-hydroxy-6-((S)-2-meth-
oxy-2-oxo-1-((1R,2S)-2-(2,2,2-trifluoroethanamido)cyclopen-
tanecarboxamido)ethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropy-
rimidin-1(2H)-yl)tetrahydro-2H-pyran-4-yl)ethane-1,2-diyl Di-
ethanoate (26). Step 1: To a room temperature solution of the
nucleoside 31 (0.034 g, 0.05 mmol) in anhydrous EtOAc (3 mL)
was added 10% palladium on activated carbon (0.035 g), then the
mixture was stirred under an H2 atmosphere for 2 h. The reaction
mixture was filtered through Celite, and the residue was washed
with EtOAc and MeOH. The filtrate was concentrated to yield the
free amine as a white solid (0.27 g), which was taken on to the
next step without further purification.
organic layer was dried (Na2SO4) then concentrated, and the residue
was purified by flash chromatography (EtOAc/hexanes ) 4:1 to
9:1) to afford the amide 26 as a white solid (0.025 g, 68%): mp
128-130 °C; [R]D -23.31 (c 0.8, CHCl3); IR (NaCl) 3275, 1740,
1
1707, 1654 cm-1; H NMR (500 MHz, CDCl3) δ 1.60-1.67 (m,
1H), 1.78-1.86 (m, 5H), 1.91-1.99 (m, 2H), 2.02-2.10 (m, 11H),
2.15-2.20 (m, 1H), 2.30-2.38 (m, 1H), 3.02-3.11 (m, 1H), 3.88
(s, 3H), 4.03-4.10 (m, 1H), 4.13 (2 d, J ) 6.6 and 11.9 Hz, 1H),
4.53 (br s, 1H), 4.57 (dd, J ) 3.3 and 12.1 Hz, 1H), 4.80 (d, J )
7.7 Hz, 1H), 4.99 (br s, 1H), 5.42 (d, J ) 4.6 Hz, 1H), 5.47 (br s,
1H), 7.17 (s, 1H), 7.82 (br s, 1H), 8.64 (br s, 1H), 9.23 (br s, 1H);
13C NMR (201 MHz, CDCl3) δ 12.6, 20.7, 21.0, 21.1, 22.9, 29.2,
29.9, 32.7, 46.5, 53.1, 53.6, 55.4, 62.6, 71.8, 73.2, 74.4, 111.8,
114.0, 115.4, 116.8, 118.2, 150.4, 156.7, 156.8, 157.0, 157.2, 163.4,
169.7, 170.0, 170.3, 171.3, 174.3; HRMS calcd for C29H38F3N4O14
m/z (M + H)+ 723.2337, found 723.2335.
“Thymine Amipurimycin” Hydrochloride (27). To an ice-
cooled solution of the protected nucleoside 26 (0.016 g, 0.02 mmol)
in anhydrous THF (2.4 mL) was added a solution of LiOH ·H2O
(0.007 g, 0.18 mmol) in water (0.6 mL). After being stirred at 0
°C for 5 h the reaction was carefully acidified to pH 3 with 10%
aq HCl at 0 °C. The solvent was removed under high vacuum and
the residue was triturated with diethyl ether (3 × 2 mL) to afford
“thymine amipurimycin” 27 as a white semisolid (0.011 g,
quantitative): [R]D 6.3 (c 0.55, H2O); IR (NaCl) 3393, 1705, 1684,
1647 cm-1 1H NMR (400 MHz, D2O) δ 1.70-2.00 (m, 8H),
;
2.11-2.17 (m, 3H), 3.08 (q, J ) 6.5 Hz, 1H), 3.71 and 3.74 (2 d,
J ) 6.8 and 11.8 Hz, 1H), 3.78-3.89 (m, 3H), 4.34-4.39 (m, 2H),
4.52 (d, J ) 3.7 Hz, 1H), 5.92 (d, J ) 9.6 Hz, 1H), 7.57 (s, 1H);
13C NMR (201 MHz, D2O) δ 11.5, 21.4, 28.2, 30.2, 36.1, 45.3,
53.5, 56.1, 61.6, 72.4, 72.6, 72.8, 76.7, 81.7, 112.1, 137.2, 152.2,
166.2, 172.2, 174.8; HRMS calcd for C20H31N4O10 (free amine)
m/z (M + H)+ 487.2040, found 487.2015.
Acknowledgment. We thank the Herman Frasch Foundation
(American Chemical Society) for financial support. Predoctoral
research fellowships to C.S.S., from the American Chemical
Society Medicinal Chemistry Division (2004-2005) (sponsored
by Aventis), and the American Foundation for Pharmaceutical
Education (2005-2006), are also gratefully acknowledged.
Step 2: To a stirring solution of N-TFA-(1R,2S)-cispentacin 254d
(0.02 g, 0.079 mmol) in anhydrous THF (2 mL) were added N,N-
diisopropylethylamine (22 µL, 0.13 mmol) and DEPBT (0.039 g,
0.13 mmol). The reaction was stirred at rt for 30 min, and then the
amine (0.027 g, 0.05 mmol), obtained from step 1 above, and
dissolved in anhydrous THF (2 mL), was added to the preactivated
acid. The reaction mixture was stirred at rt for 12 h, and then was
poured into a separatory funnel and diluted with EtOAc (5 mL).
The organic layer was washed successively with 5% NaHSO4 (1
× 5 mL), brine (1 × 5 mL), and 10% Na2CO3 (1 × 5 mL). The
Supporting Information Available: General experimental
details and copies of NMR spectra (1H and 13C) of all the new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO8004815
4174 J. Org. Chem. Vol. 73, No. 11, 2008