Synthesis of an apionucleoside family and discovery of a prodrug with anti-HIV activity

Article abstract of DOI:10.1021/jo500659e

We report the synthesis of a family of d- and l-furano-d-apionucleosides, their 3'-deoxy, as well as their 2',3'-dideoxy analogues with thymine and adenine nucleobases. Single carbon homologation of 1,2-O-isopropylidene-d- glycero-tetrafuranos-3-ulose (15) and optimized glycosylation conditions involving microwave irradiation were key to the successful synthesis of the target compounds. While all target nucleosides failed to show significant antiviral activity, we demonstrated that the triphosphate of 2',3'-deoxy-d-apio-d-furanoadenosine (1), in contrast to that of its d-apio-l-furanose epimer 2, was readily incorporated into a DNA template by HIV reverse transcriptase to act as a DNA chain terminator. This led us to convert adenine derivative 1 into two phosphoramidate prodrugs. ProTide 9b was found active against HIV-1 and HIV-2 (EC₅₀ = 0.5-1.5 μM), indicating that the lack of activity of the parent nucleoside, and possibly also other members of the d-apio-d-furanose nucleoside family must be sought in the inefficient cellular conversion to the monophosphate.

Full text of DOI:10.1021/jo500659e

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Article
Synthesis of an apionucleoside family and
discovery of a prodrug with anti-HIV activity.
Kiran S Toti, Marco Derudas, Fabrizio Pertusati, Davy Sinnaeve, Freya Van den Broeck, Lia Margamuljana,
Jose C. Martins, Piet Herdewijn, Jan Balzarini, Christopher McGuigan, and Serge Van Calenbergh
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo500659e • Publication Date (Web): 07 May 2014
Downloaded from http://pubs.acs.org on May 15, 2014
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Synthesis of an apionucleoside family and discovery of a
prodrug with anti-HIV activity
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a
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Kiran S. Toti, Marco Derudas, Fabrizio Pertusati, Davy Sinnaeve, Freya Van den
c
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Broeck, Lia Margamuljana, José C. Martins, Piet Herdewijn, Jan Balzarini, Christopher
b
a,
McGuigan, Serge Van Calenbergh *
a
Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent
University, Harelbekestraat 72, B-9000 Ghent, Belgium.
b
Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood
Building, King Edward VII Avenue, Cardiff CF10 3NB, UK.
c
NMR and Structure Analysis Unit, Department of Organic Chemistry, Ghent University,
Krijgslaan 281 S4, B-9000 Ghent, Belgium.
d
Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium.
*
Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, UGent,
Harelbekestraat 72, B-9000 Gent, Belgium. Phone: +32 9 264 81 24. Fax: + 32 9 264 81 46.
E-mail: serge.vancalenbergh@ugent.be
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Graphical Abstract
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Abstract
We report the synthesis of a family of D- and L-furano-D-apionucleosides, their 3’-
deoxy-, as well as their 2’,3’-dideoxy-analogues with thymine and adenine nucleobases.
Single carbon homologation of 1,2-O-isopropylidene-D-glycero-tetrafuranos-3-ulose (15) and
optimized glycosylation conditions involving microwave irradiation were key to the
successful synthesis of the target compounds.
While all target nucleosides failed to show significant antiviral activity, we
demonstrated that the triphosphate of 2’,3’-deoxy-D-apio-D-furanoadenosine (1), in contrast
to that of its D-apio-L-furanose epimer 2, was readily incorporated into a DNA template by
HIV reverse transcriptase to act as a DNA chain terminator. This led us to convert adenine
derivative 1 into two phosphoramidate prodrugs. ProTide 9b was found active against HIV-1
and HIV-2 (EC = 0.5-1.5 µM), indicating that the lack of activity of the parent nucleoside
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and possibly also other members of the D-apio-D-furanose nucleoside family must be sought
in the inefficient cellular conversion to the monophosphate.
Keywords: Apionucleosides, ProTides, antivirals, RT inhibitor, microwave synthesis,
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nucleoside triphosphate.
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Introduction
Although the pharmacological scope of nucleoside analogues is still expanding, they
remain most renowned for their utility as antiviral drugs [1]. 2’,3’-Dideoxy-β-D-apio-D-
furanonucleosides (D-ddANs, 1, Figure 1) were synthesized in the early 1990s as potential
antiviral agents, but were found inactive [2,3,4]. However, some of us recently discovered
that the 3’-O-phosphonomethylated adenine (A) and thymine (T) analogues 7 exhibit
promising anti-HIV properties [5]. Since these phosphonates act as bioisosteres of the
phosphorylated species 8, we decided to reinvestigate the biological activity of these ddANs.
We envisioned a synthetic approach that would also give access to the known
apionucleosides 3 [6,7], their 3’-deoxy counterparts 2 [8,9,10,11], and inadvertently also the
D-apio-L-furanose epimers 4-6. Furthermore, we planned to expand the potential of the 2’,3’-
dideoxyapio nucleosides 1 and 4 as antiviral agents by synthesizing their phosphoramidate
prodrugs 9, 10 and 11. These would lead to the intracellular release of the parent nucleotides
like 8 [12, 13], thereby by-passing the often problematic first phosphorylation step in the
conversion to the active triphosphate species [14].
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Figure 1.
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Results and Discussion
Chemistry
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Scheme 1. Synthesis of the D- and L-furano-D-apiose coupling partners 17 and 24 and their 3-deoxy
analogues 19, 20 and 28. Reagents and conditions: (a) TEMPO, BAIB, CH Cl , rt, 3-4h, 90%; (b)
2
2
BOMSnBu , n-BuLi, THF, -78 °C, 2h, 68%; (c) (i) 80% aq. AcOH, 80 °C, 8h; (ii) Ac O, DMAP,
3
2
pyridine, 55 °C, 16h, 75%; (d) (i) NaH, CS , MeI, THF, 0 °C → rt, 1h; (ii) Et B, Bu SnH, toluene, rt,
2
3
3
3-4h, 68%; (e) (i) 80% aq. AcOH, 80 °C, 8h; (ii) Ac O, DMAP, rt, pyridine, 4h, 57%; (f) (i) p-TsOH
2
(para-toluenesulfonic acid), MeOH, rt, overnight; (ii) Ac O, DMAP, pyridine, 0 °C → rt, 4h, 77%;
2
(
g) CH COOH-H O (2:1), rt, 3 days, 83%; (h) Bu SnO, toluene, 140 °C, 2h, TBAB, BnBr, 100 °C,
3 2 2
1
8h, 94%; (i) H , Pd/C, MeOH, rt, 5h, 90%; (j) acetone, conc. H SO , rt, 1.5h, Na CO , 45 min, 73%
2 2 4 2 3
(
after 3 cycles); (k) DMF, NaH, 0 °C, 10 min, BnBr, 0 °C → rt, 18h, 95%; (l) (i) 80% aq. AcOH,
80 °C, 18h; (ii) pyridine, Ac O, rt, 18h, 79%.
2
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Compounds 16 and 27 were considered valuable intermediates to access the envisaged family
of D-furanoapionucleosides (Scheme 1). They were prepared from 1,2-isopropylidene-α-L-
threose (14), which was obtained in six steps from L-ascorbic acid [15,16]. Interestingly,
screening of different oxidation methods [15,17,18] to convert 14 to ketone 15 indicated that
TEMPO-BAIB ([Bis-(acetoxy)-iodo]benzene) oxidation, best known for oxidation of primary
hydroxyl groups, was the most effective. Conversion of 15 to 16 is feasible by reacting the
former with diazomethane to give a spiro-oxirane [19], which can then be opened with
benzylalkoxide to give 16 [20]. To avoid the use of diazomethane, we explored several
variations of the polarity reversal concept to realize the desired carbon homologation.
Reaction with benzyloxymethyl chloride in the presence of samarium iodide did not yield the
desired product, while the corresponding Grignard reaction gave 16 in disappointing yields
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[21]. Nucleophilic attack of the ketone with lithiated benzyloxymethyltributyltin afforded 16
in acceptable yield [22], considering the propensity of compound 15 to undergo self-
condensation to the aldol dimer [23]. The NMR spectra of 16 were in accordance with
reported data [20] and C-3 stereochemistry was further confirmed by a two-dimensional (2D)
1
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H- H NOESY experiment. One-pot acid hydrolysis and acetylation of 16 gave the tri-
acetylated apiose 17 in a 2:1 α/β anomeric ratio.
Jin et al. reported the conversion of 23 to 27 using Barton-McCombie deoxygenation
(BMD) [18]. Surprisingly, BMD of 16 afforded 18 instead of 27. This led us to reinvestigate
the protocol of Jin et al. on compound 23, prepared from the commercially available 21 [24].
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In our hands BMD on 23 also gave 18. The stereochemistry was confirmed by 2D H- H
NOESY experiment and by independent synthesis of compound 27. The formation of 18 is
explained by radical quenching from the least hindered face, i.e. opposite to the
isopropylidene comprising face [ 25 ]. Furthermore, Jin et al. probably synthesized the
enantiomer of 18, since they started from D-galactose, which should lead to 1,2-O-
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isopropylidene-α-D-threofuranose (i.e., the enantiomer of 14 [15]). Compound 23 was
hydrolyzed and acetylated to give the L-furano analogue of triacetylated apiose 24.
Compound 18 was hydrolyzed and acetylated to give 19 in a 4:1 (β:α) anomeric ratio.
The anomeric configuration was inferred from the anomeric proton coupling constants, i.e. 0
Hz for the β-isomer and 4.4 Hz for the α-isomer. However, this conversion lacked
reproducibility, especially on a larger scale. To overcome this problem, the methyl anomer 20
was synthesized in two steps from 18. Since the coupling constant for anomeric hydrogen is
close to 0 Hz, 20 is assumed to be the β-isomer.
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Carey and co-workers found that 1,2-O-isopropylidine-L-furano-D-apiose 22 equilibrates
into a mixture of the D- and L-furanose form in acidic acetone, which inspired us to use
similar conditions for the epimerization of 25 [19]. We hypothesized that the absence of the
3
-hydroxyl group would eliminate the repulsive dipole interaction with oxygen at position 2,
while the steric interaction of the hydroxymethyl group with the 2-oxygen could result in a
favorable D-furano isomer ratio. Hence compound 18 was debenzylated and then equilibrated
in acetone-conc.H SO to isolate the desired compound 26 in 73% yield after 3 equilibrium
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4
cycles. Benzylation of 26 gave 27, which upon hydrolysis and acetylation rendered 3-deoxy-
D-apio-D-furanose derivative 28 in good yields.
Having the coupling synthons 17, 19, 20, 24 and 28 in hand, we set out different coupling
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reaction conditions for 19 and 20 with silylated thymine or N -benzoyladenine under
Vorbrüggen conditions (Table 1).
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Table 1. Vorbrüggen coupling conditions.
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Entry sugar silylated
nucleobase
reagent
conditions
product
(yield)
29 (quant)
a
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19
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20
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T
TMSOTf
TMSOTf
1,2-(CH ) Cl , rt, 4h
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b
N -BzA
1,2-(CH ) Cl , 40 °C, 48h
30 (32%)
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T
TMSOTf 1,2-(CH ) Cl or CH CN, rt, 4h 32
2 2 2 3
c
SnCl4
SnCl4 d
TMSOTf
CH CN, rt, 4h
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N -BzA
CH CN, rt, 4h
-
3
T
CH CN, 150 °C, 5 min.
microwave
CH CN, 150 °C, 5 min.
29 + β-anomer
3
e
(78%)
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d
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20
N -BzA
TMSOTf
30 (60 %)
3
microwave
a
b
c
d
e
isolated yields, “-“ indicates an unresolvable reaction mixture.
the 2’-acetyl analogue of 31 was isolated in equal amount.
two diastereomers observed by TLC and HRMS analysis.
0
.2 equivalents of TMSOTf.
inseparable 2:1 mixture of 29 and its β-isomer.
Whereas the acetate anomer 19 reacted smoothly at room temperature in 4h with silylated
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thymine in the presence of TMSOTf to quantitatively give 29, coupling with N -
benzoyladenine only afforded the desired coupling product 30 in 32% yield by heating the
°
reaction mixture at 40 C for 48h [26]. This low yield resulted from the formation of an equal
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amount of an unknown isomer. H-NMR of this isomer suffered from peak broadening and
1
3
indicated the presence of minor impurities. Its UV (λmax = 331.9 nm) and C-spectrum was
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characteristic of an N -isomer [27,28]. After treatment with methanolic ammonia for two
days, a product was formed that was confidently identified as 31 (Figure 2). The binding
topology of the adenine base to the sugar was determined by NMR. A correlation between H-
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1
’and C-2 in a 2D H- C HMBC spectrum indicates that the adenine is either bound via N-1
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or N-3. In a 2D H- H NOESY spectrum, nOe cross-peaks were detected between the amide
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proton and several protons of the sugar moiety, most notably H-1’, H-2’ and ortho-protons of
the benzoyl group. In addition to this, ortho-protons of the benzoyl moiety also showed nOe
interactions with all up (α-face) protons of the furanose ring. These nOe’s are improbable if
the base is attached via N-3, since in this case the amide group and the sugar moiety would be
positioned para relative to one another and be spatially too far apart.
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Coupling reaction between methylglycoside 20 and silylated thymine (entries 3 and 4),
using either TMSOTf or SnCl , resulted in the formation of two main products that gave
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spots with comparable intensity on TLC. ESI-HRMS analysis allowed identifying these
products as the two diastereomers of 32 [18, 29 ]. The condensation reaction of
methylglycoside 20 with silylated benzoyladenine in the presence of anhydrous SnCl gave
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an unresolvable reaction mixture.
Figure 2. Byproducts (or their deprotected form) formed during Vorbrüggen coupling
Vorbrüggen coupling of the methyl anomer 20 and silylated thymine under microwave
irradiation resulted in an inseparable mixture of two isomeric products in a 2:1 ratio [30],
1
even after removal of the acetyl and benzyl protecting groups. The H-NMR spectrum of the
minor isomer 33 showed a larger splitting of the anomeric hydrogen doublet (3.2 Hz)
compared to the major compound (2.0 Hz), indicating a β-oriented pyrimidine moiety. The
gHMBC confirmed the C1’-N1 attachment, while 2D NOESY ratified the relative
stereochemistry.
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Conversely, microwave-assisted coupling between 20 and silylated N -benzoyladenine
gave only the desired α-nucleoside 30 in 60% isolated yield. Clearly, the microwave-assisted
coupling with the methyl glycoside is the method of choice to prepare the adenine nucleoside.
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Scheme 2. Synthesis of α-L-furano-D-apionucleosides 6a,b and their 3’-deoxy counterparts 5a,b.
Reagents and conditions: (a) appropriate silylated base, 1,2-(CH ) Cl , TMSOTf, rt, 4h, 85% for 34;
2
2
2
(b) appropriate silylated base, CH CN, 0.2 eq. TMSOTf, microwave (MW) 300W, 0 °C→150 °C, 3
3
min, 150 °C, 5 min, 40% for 35 and 6% for 36; (c) NH , MeOH, rt, 4-48h, 75-96%; (d) H , Pd/C,
3
2
MeOH, rt, overnight, 86% for 5a from 37 and 71% for 6a from 39;(e) (i) Pd(OH) , HCOOH-MeOH
2
(1:1 for 5b, 41 from 38/1:9 for 6b from 40), 55 °C, 5-8h; (ii) NH , MeOH, rt, 3h, 80% over two steps
3
for 5b and 6b.
Reaction of the triacetyl apiose 24 with silylated thymine under classical Vorbrüggen
conditions provided 34 in very good yield (Scheme 2). Microwave conditions were employed
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to couple 24 with silylated N -benzoyladenine, affording 35 and minor amounts of the 2’-
OTMS analogue 36. The coupling products 29, 30, 34 and 35 were treated with ammonia in
methanol to provide the desired deacetylated products 37-40. Debenzylation of L-furano-D-
apio thymine nucleosides 37 and 39 to give the 3’-deoxyapionucleoside 5a and
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apionucleoside 6a was realized by Pd-catalyzed hydrogenation. The same reaction condition
on adenosines 38 and 40 was ineffective, as well as the use of cyclohexene and ammonium
formate. This led us to use formic acid as hydrogen source to give 5b and 6b. The byproduct
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1 was converted to 5b upon treatment with ammonia in methanol.
Scheme 3. Synthesis of β-D-furano-D-apionucleosides. Reagents and conditions: (a) silylated thymine,
6
1
1
,2-(CH ) Cl , TMSOTf, rt, 4h; (b) silylated N -BzA, CH CN, 0.2 eq. TMSOTf, MW 300W, 0 °C →
2 2
2
3
50 °C, 3 min, 150 °C, 5 min; (c) 7N NH -MeOH, rt, 4-48h, 46-97% over two steps, 28% for 43 and
3
11% of its α-anomer; (d) for 42 and 48, H , Pd/C, MeOH, rt, 4h, 86-89%; (e) (i) Pd(OH) , HCOOH-
2
2
MeOH (1:4), 55 °C, 5h (ii) NH , MeOH, rt, 3h, 89% for 2b and 3b; (f) thiocarbonyl diimidazole,
3
DMF, 80 °C, 90 min, 89% for 46 and 78% for 47; (g) P(OCH ) , 120 °C, 6h, 90%.
3
3
Using similar protocols 17 and 28 were converted to 2a,b and 3b in acceptable yields
(Scheme 3). Compared to the L-series, this sequence gave low yields for both thymine and
adenine analogues, in particular for the 3’-deoxy analogues. Moreover, coupling of 28 with
silylated benzoyladenine produced significant amount of α-isomer (11%), possibly due to
participation of the 3’-benzyloxymethyl group, with only 28% of desired β-nucleoside. Since
the synthesis of 1a,b via 28 involves many linear steps, we envisaged more convenient access
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to 2’,3’-dideoxy-β-D-apio-D-furanonucleosides 1a,b involving Corey-Winter olefination [31]
and stereoselective hydrogenation as key steps. During catalytic hydrogenation the syn-
addition of the hydrogen atoms to the double bond is anticipated to occur from the face
opposite to the nucleobase [32]. Thiocarbonylation of 44 and 45 using thiocarbonylimidazole
provided precursor compounds 46 and 47 for Corey-Winter olefination. Unfortunately, the
adenine derivative degraded in trimethylphosphite at 120 °C. The thymine derivative gave the
desired product 48 in excellent yield but the hydrogenation reaction resulted in a mixture of
diastereomers 1a and 4a that were inseparable by column chromatography. This forced us to
follow the classical route via 2a,b to the target 2’,3’-dideoxy analogues.
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Initially, the benzyl protected nucleosides 37 and 38 were subjected to a standard
Barton-McCombie protocol to give the 2’-deoxygenated products 53 and 54 (Scheme 4).
Different hydrogen sources were explored for the subsequent Pd-catalyzed debenzylation, but
only the thymine compound 53 could be converted to the desired product 4a with curtailed
reproducibility. This was attributed to catalyst poisoning by remaining sulfur residues. Hence,
we swapped to TBS as a protecting group to give 49-52 from 5a,b and 2a,b in excellent
yields. Compounds 49-52 were submitted to BMD after conversion to the corresponding
xanthates with p-tolylchlorothionoformate in the presence of DMAP. These xanthates were
isolated after a brief workup and heated in toluene with tributyltin hydride and
azobisisobutyronitrile to give the 2’,3’-dideoxyapiose nucleosides 55-58. The TBS group of
5
5 and 56 was removed using TBAF in THF. However, the removal of the
tetrabutylammonium salt to get pure adenosine derivative 4b was not satisfactory, hence we
used NH F in methanol at 55 °C for 2 days to give 4a,b and 1a,b in excellent isolated yields
4
[33].
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Scheme 4. Synthesis of D- and L-furano-D-dideoxydihydroapionucleosides. Reagents and conditions:
(
a) TBSCl, imidazole, DMF, rt, overnight, 82-95%; (b) (i) p-tolylchlorothionoformate, DMAP, ACN,
°C → rt, 4h; (ii) Bu SnH, AIBN, toluene, reflux, 2-3h, 70-90% over two steps; (c) H /Pd-C,
0
3
2
methanol, rt, overnight, 53 to 4a, 63%; (d) TBAF, THF, rt, 3h, 55 to 4a, 89%; (e) NH F, MeOH,
4
50 °C, 2 days, 86-94%.
To examine the potential of their monophosphate prodrugs as anti-HIV agents, apio-
dideoxynucleosides 1b and 4b were converted to the corresponding triphosphates 12 and 13,
following the method of Caton-Williams (Scheme 5) [34]. The yield of the D-isomer 12 was
1
31
low and H NMR indicated internal salt formation. Likewise, the P NMR of this compound
is uncharacteristic of triphosphate salts, as it showed two broad peaks. The addition of two
equivalents of triethylamine disrupted this internal salt leading to the appearance of the
characteristic triphosphate peaks (See supporting information).
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Scheme 5. Synthesis of D- and L-furano-D-dideoxyapioadenosine triphosphates (D-/L-
ddAATP) 12 and 13. Reagents and conditions: (a) 59, 60, n-Bu N, anh. DMF, rt, 1.5h; (b) (i)
3
1
b/4b, anh. DMF, rt, 1.5h; (ii) I , rt, 20 min, H O, rt, 1.5h, 21% for 12 and 48% for 13.
2 2
Nucleoside monophosphate prodrugs (ProTides), featuring an alanine as the preferred
amino acid [35,36,37], were prepared using two different methods (Scheme 6) [38]. The
thymine analogue 11a was prepared by coupling 4a with the phosphorochloridate 64a, using
tert-butylmagnesium chloride as hydroxyl activator. Under similar reaction conditions
compound 4b degraded. Hence, all other analogues were coupled with 64a/b using N-
methylimidazole (NMI) as a base in a mixture of anhydrous THF and pyridine as solvents. In
all cases, the desired compounds were obtained as a mixture of two diasteroisomers resulting
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from the two possible configurations of the phosphorous stereo center, as confirmed by the
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presence of two equal height peaks in the P-NMR spectrum.
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Scheme 6. Synthesis of apionucleoside ProTides. Reagents and conditions: (a) CH Cl , TEA, -78 °C,
2
2
30 min, rt, 2h, 87-96%; (b) t-butyl magnesium chloride, THF, rt, overnight, 22% for 11a; (c) NMI,
THF, pyridine, rt, 2 days, 15-88%.
DNA Chain termination study using HIV Reverse Transcriptase
A prerequisite for ProTides to show a good biological profile is that the corresponding
triphosphates are good substartes for the final target, such as reverse transcriptase (RT) for
HIV. Hence we investigated the ability of triphosphates 12 and 13 to act as a substrate of
HIV-RT in a primer-template assay [39]. The template has overhanging T residues to test
incorporation of the modified A nucleotide. Figure 3 clearly shows that both nucleotides 12
and 13 function as DNA chain terminators. The D-furano analogue 12 is more efficiently
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incorporated than its 3’-epimer 13, but compared to natural substrate 2’-deoxyadenosine
triphosphate (dATP), requires a higher concentration and longer time for complete
incorporation. The characteristics of 12 towards HIV RT render the corresponding ProTides
as potentially useful HIV inhibitors.
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Figure 3. Electrophoregram that shows DNA chain termination through incorporation of
dideoxydihydro-D-apio-D-furano-adenosine triphosphate (12) and dideoxydihydro-D-apio-L-
furano-adenosine triphosphate (13) by HIV RT. The DNA polymeration mixtures containing 125
nM annealed (labeled) primer-template complex, were treated with 125, 500, or 1000 µM of modified
-1
triphosphate (12/13) and 0.03 U.µl HIV RT and incubated at 37°C. Aliquots were taken after 15, 30
and 60 min. In the control reaction, 50 µM of natural dATP was used. Samples were separated on a
0.4 mm 20% denaturing polyacrylamide gel and the bands visualized using phosphorimaging.
Enzymatic assay using carboxypeptidase Y
The putative mechanism of activation of ProTides [40, 41, 42] (Figure 4) involves an
enzymatic cleavage of the ester (step a) mediated by an esterase- or carboxypeptidase-type
enzyme followed by spontaneous cyclisation with releasing the phenolate anion (step b) and
to open the unstable mixed anhydride ring by water (step c) providing the intermediate
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metabolite (D / L) 67a/b. The cleavage of the phosphorous-nitrogen bond of the latter (step d)
requires a phosphoramidase-type enzyme, perhaps related to human HINT-1, to release the
monophosphate form (D- / L- 68 a/b).
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Figure 4. Putative mechanism of bioactivation for monophosphate prodrugs.
In order to investigate this mechanism of bioactivation for ProTides 9a and 11a,b, we
performed an enzymatic experiment incubating the compounds with carboxypeptidase Y
3
1
enzyme in acetone-d and Trizma buffer (pH = 7.6) recording a P-NMR at specific time
6
intervals. The L-furano series displayed pronounced difference in rate of hydrolysis among
two diastereomers, a phenomenon that was observed earlier for ProTides of phosphates [38]
3
1
and phosphonates [43]. For instance, one of the diastereoisomer of 11a ( P-NMR = 3.3 ppm,
Figure 5, panel A) seems to be more slowly converted compared to the other. In fact, after
1
8h, it is still present, even after the addition of an extra portion of enzyme, while the
diastereomer at 3.5 ppm appears fully converted after about 10 minutes. In contrast,
31
compound 11b ( P-NMR = 3.2 and 3.4 ppm, Figure 5, panel B) shows a near complete
3
1
conversion of both diasteroisomers to the metabolite L-67b ( P-NMR = 7.0 ppm) through
31
the intermediate L-65b ( P-NMR = 4.5 ppm) after 1 hour, although there again exists a
clear difference in kinetics.
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11a
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L-65a
L-67a
11b
L-65b
L-67b
3
1
Figure 5. P-NMR stack spectra for bioactivation study of compounds 11a (panel A) and 11b (panel
B) using carboxypeptidase Y enzyme. The assignment of the resonance signals to the indicated
metabolites was done in analogy with previous studies [35].
3
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Within 20 minutes after addition of the enzyme compound 9a ( P NMR = 3.5 and 3.7
3
1
ppm) was completely converted to the intermediate metabolite D-65a ( P-NMR = 4.5 and 4.8
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ppm), which was fully converted to compound D-67a ( P-NMR = 7.1 ppm) within an hour
(Figure 6). In this case no pronounced diastereomeric discrimination by carboxypeptidase
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enzyme was observed. Following the trend for adenine analogue 11b, we assume that 9b
would be processed at the least with the rate of thymine analogue 9a.
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D-67a
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a
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Figure 6. P-NMR stack spectra for bioactivation study of compound 9a using carboxypeptidase Y
enzyme. The assignment of the resonance signals to metabolites D-65a and D-67a is based on LC-MS
experiments.
From this study it is evident that both D- and L-furanonucleoside ProTides are readily
converted to the intermediate metabolite 67.
Biological Evaluation
The 2’,3’-dideoxy analogues 1a,b and the 3’-deoxy-β-D-apio-D-furanonucleosides
2
a,b failed to show both activity against HIV-1,2 and cytotoxicity. Likewise, the 2’,3’-
dideoxy analogues 4a,b and the 3’-deoxy-β-D-apio-L-furanonucleosides 5a,b lacked
significant activity against HIV-1 and HIV-2 and a panel of other DNA and RNA viruses,
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and were also devoid of cytotoxicity. The thymine-based ProTides 9a and 10a were also
devoid of anti-HIV activity, which might be due to inefficient conversion of the alaninyl d-
ddATMP to the corresponding monophosphate by HINT-1-type phosphoramidase enzyme or
further kinase mediated conversion to the corresponding triphosphate. Alternatively, the latter
may be inefficiently incorporated by HIV RT (Table 2).
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Interestingly, the 2’,3’-dideoxy-D-apio-D-furanoadenosine phosphoramidate ProTides
9
b and 10b combine potent and moderate anti-HIV activity with reasonable selectivity. The
benzylester 9b exhibits comparable or even somewhat superior anti-HIV activity to the
acyclic nucleoside phosphonate R-PMPA (tenofovir) [44]. The ProTides 9a,b, 10a,b and
1
1a,b are weak to moderate inhibitors of murine leukemia (L1210), human T-lymphocyte
(CEM) and human cervix carcinoma (HeLa) cell proliferation (Table 3).
Table 2. Antiviral activity and cytotoxicity of ProTides 9a,b and 10a,b
EC in MT-4 cells (µM)
EC in CEM cells (µM)
50
50
HIV-1
HIV-2 (ROD) CC50
HIV-1 (IIIb) HIV-2 (ROD)
(
NL4.3)
>250
>250
0.5
9
10a
a
>250
>250
1.0
24
1.0
196
>250
93
>250
>250
-
-
0.5
7.5
3.0
-
-
1.5
38
2.5
9
b
1
0b
R-PMPA
-‘ = not performed
26
1.7
‘
a
Table 3. Cytotoxicity data of ProTides 9a,b and 10a,b
L1210
113 ± 21
110 ± 17
> 250
226 ± 35
167 ± 85
79 ± 4
CEM
108 ± 11
80 ± 4
> 250
204 ± 3
HeLa
159 ± 32
53 ± 11
> 250
≥ 250
177 ± 103
173 ± 58
9
a
9
b
1
1
1
1
0a
0b
1a
1b
113 ± 3
73 ± 5
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a
IC in µM, murine leukemia cells (L1210/0), human T-lymphocyte cells
CEM/0) and human cervix carcinoma cells (HeLa)
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(
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Conclusion
In this study we report the synthesis of a family of D-apionucleosides comprising the A and T
members of both possible 3’-epimers of β-D-apiofuranose nucleosides, as well as their 3’-
deoxy and 2’,3’-dideoxy analogues. Clues in the synthesis of the desired apionucleosides
were a carbon homologation of 1,2-O-isopropylidene-D-glycero-tetrafuranos-3-ulose (15) and
optimized glycosylation conditions involving microwave irradiation. In the course of this
work, we rectified some anomalies in the structure assignments reported by others.
In accordance with earlier reports the target D-apio-D-furanose nucleosides failed to show
antiviral activity and so did their D-apio-L-furanose epimers. However, the triphosphate of
2
’,3’-dideoxy-β-D-apio-D-furanoadenosine (12) (in contrast to its D-apio-L-furanose epimer
3) was readily accepted by viral DNA polymerase to act as a DNA chain terminator. This
1
led us to convert the parent A and T nucleosides 1a and 1b into phosphoramidate prodrugs 9
and 10. The A analogues 9b and 10b indeed showed a considerable anti-HIV activity. This
indicates that the lack of activity of the parent 2’,3’-dideoxy-β-D-apio-D-furanose nucleoside
must be the result of inefficient conversion to the monophosphate in the biological assay.
This study demonstrates that the large pool of nucleoside analogues that were previously
found to lack antiviral activity may contain valuable candidates to be turned into ProTide
derivatives exhibiting promising antiviral activity, by efficiently bypassing the first
phosphorylation step that is often rate-limiting the intracellular conversion of nucleoside
analogues to their bio-active triphosphate derivatives.
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Experimental Section
Synthesis
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All reagents were from standard commercial sources and of analytic grade. Dry
solvents were obtained directly from commercial sources and stored on molecular sieves. All
reactions were carried out under argon atmosphere using anhydrous solvents unless specified
otherwise. Room temperature or rt refers to 25±5 °C. Silica-gel precoated with F254 plates
were used for TLC. The spots were examined under ultraviolet light at 254 nm and further
visualized by sulphuric acid-anisaldehyde spray. Column chromatography was performed on
silica gel (40-63 µm, 60 Å) or on flash chromatography system. NMR spectra were recorded
on a 300 MHz, 500 MHz or 700 MHz spectrometer. Chemical shifts are given in ppm (δ),
calibrated to the residual solvent signals or TMS. Exact mass measurements were performed
on mass spectrometer equipped with a standard electrospray ionization (ESI) and modular
LockSpray TM interface. Samples were infused in a CH CN/H O (1:1v/v) mixture at 10
3
2
mL/min. The microwave reactions were carried out in Milestone MicroSYNTH Advanced
Microwave Synthesis Labstation, equipped with 2 X 800 W magnetrons (effective maximum
output 1500W pulsed/continuous), an optical fiber temperature sensor, a pressure sensor, in
continues power mode in a closed PTFE vessel. NMR signals of sugar protons and carbons
are indicated with a prime, and signals of base protons and carbons are given without a prime.
1
13
A combination of gCOSY, gHSQC and gHMBC was used to assign H and C peaks, Noesy
was used for selected compounds to assign peaks and/or to confirm relative configuration.
[
18]
3
-Oxo-1,2-O-isopropylidene-α-D-erythrofuranose (15) : To a solution of compound 14
(
1.0 g, 6.24 mmol) in CH Cl (12.5 mL) was added bis-acetoxyiodobenzene (BAIB, 2.41 g,
2
2
7
.5 mmol) followed by (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO, 195 mg, 1.25
mmol) at room temperature under an argon atmosphere. The mixture was stirred at room
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temperature for 4h. The contents of the reaction was directly loaded on silica-gel and eluted
1
with 30% EtOAc-hexanes to afford pure product 15 (890 mg, 90%) as a white solid. H NMR
(
300 MHz, CDCl ) δ ppm 1.35 (s, 3H, CH ), 1.44 (s, 3H, CH ), 4.03 (dd, J = 4.1, 17.6 Hz,
3 3 3
0
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H, 4-H), 4.29 (s, 1H, 2-H), 4.32 (dd, J = 0.6, 17.6 Hz, 1H, 4-H), 6.02 (d, J = 4.4 Hz, 1H, 1-
H).
[
20]
1
,2-O-Isopropylidene-5-(O-benzyl)-α-D-apio-D-furanose (16) : To a stirring solution of
benzyloxymethyltributlytin (BOMSnBu , 5.93 g, 14.4 mmol) in THF (35 mL) at -78 °C
3
under inert condition, was added dropwise n-butyllithium (1.6M in hexanes,19.5 mL, 31.3
mmol) and stirred for additional 1h. To this mixture was then added dropwise a solution of
compound 15 (1.9 g, 12.02 mmol) in 10 mL THF and stirred at -78 °C for 3h. The reaction
was quenched with saturated NH Cl solution and by vigorous stirring. EtOAc (100 mL) was
4
then added to facilitate the layer separation. Organic layer was separated and the aqueous
layer was extracted twice with EtOAc (50 mL). Combined organic layers were dried over
anhydrous MgSO and evaporated under reduced pressure. The residue was purified by
4
column chromatography eluting with 17% EtOAc-hexanes to afford 16 (2.3 g, 68%) as a
1
white solid. H NMR (300 MHz, CDCl ) δ ppm 1.37 (s, 3H, CH b), 1.58 (s, 3H, CH a), 2.85
3
3
3
(
s, 1H, 3-OHa), 3.46 (d, J = 10.3 Hz, 1H, 5-H), 3.56 (d, J = 10.3 Hz, 1H, 5-H), 3.71 (d, J =
.1 Hz, 1H, 4-Ha), 3.80 (d, J = 9.1 Hz, 1H, 4-Hb), 4.39 (d, J = 3.8 Hz, 1H, 2-Hb), 4.54 - 4.71
(m, 2H, CH Ph), 5.76 (d, J = 4.1 Hz, 1H, 1-Hb), 7.27 - 7.40 (m, 5H, CH Ph).
9
2
2
[45]
1
,2,3-Tri-(O-acetyl)-5-(O-benzyl)-α/β-D-apio-D-furanose (17) : A solution of 16 (2.5 g,
8
.92 mmol) in 80% aq. acetic acid (25 mL) was stirred at 80 °C for 8h. The reaction mixture
was evaporated to give the crude intermediate as syrup. This syrup was dissolved in pyridine
20 mL) and DMAP was added (100 mg) followed by acetic anhydride (10 mL, 106 mmol).
The solution was stirred at 55 °C for 16h. Then, the solvent was removed under vacuum and
(
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the resulting residue was partitioned between EtOAc and water. Organic layer separated,
combined organic layer was washed with brine, dried over sodium sulfate, and evaporated.
The residue was purified by silica-gel column chromatography (15-20% EtOAc-hexanes) to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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44
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46
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48
49
50
51
52
53
54
55
56
57
58
59
60
1
yield 17 (2.45 g, 75%) as a colorless oil as a mixture of α + β isomers (2:1). H NMR (300
MHz, CDCl ) δ ppm 1.96 (s, 3H, major), 2.08 (s, 3H, major), 2.08 (s, 2H, minor), 2.09 (s, 1H,
3
minor), 2.10 (s, 3H, major), 3.75 (d, J = 9.7 Hz, 0.47H, minor), 3.89 (d, J=10.5 Hz, 1H,
major), 3.96 (d, J = 9.7 Hz, 0.5H, minor), 4.05 (d, J = 10.5 Hz, 1H, major), 4.22 (d, J = 10.3
Hz, 1H, major), 4.26 (d, J = 10.5 Hz, 0.52H, minor), 4.32 (d, J = 10.5 Hz, 0.5H, minor), 4.34
(
d, J = 10.3 Hz, 1H, major), 4.51 - 4.62 (m, 3H, major & minor), 5.42 (d, J = 4.7 Hz, 0.44H,
minor), 5.49 (d, J = 1.2 Hz, 1H, major), 6.08 (d, J = 1.2 Hz, 1H, major), 6.33 (d, J = 4.7 Hz,
+
0.43H, minor), 7.27 - 7.41 (m, 7H, major & minor). HRMS (ESI-TOF) m/z: [M + Na] Calcd
for C H O Na 389.1212; Found 389.1242.
1
8
22
8
[18]
1,2-O-Isopropylidene-3-deoxy-5-(O-benzyl)-β-D-apio-L-furanose (18) : To a solution of
1
6 (3.5 g, 12.5 mmol) in dry THF (75 mL) was added NaH (60% in mineral oil, 1.5 g, 37.45
mmol) at 0 °C and the reaction mixture was stirred at room temperature for 1h. To this
mixture were slowly added CS (11.2 mL, 188 mmol) and MeI (24.0 mL, 375 mmol) and
2
stirred at room temperature for 1h. The reaction mixture was evaporated to give crude
xanthate. The xanthate was suspended in dry toluene (75 mL), triethylborane (19.0 mL, 19.0
mmol, 1.0 M solution in THF) and n-Bu SnH (5 mL, 19.0 mmol) were added at room
3
temperature and the mixture was stirred for further 3h. The reaction mixture was quenched
with water, extracted with EtOAc, dried over anhydrous MgSO , filtered and evaporated. The
4
residue was purified by silica gel column chromatography (10% EtOAc-hexanes) to give 18
1
(
2.26 g, 68 %) as a colorless oil. H NMR (300 MHz, CDCl ) δ ppm 1.32 (s, 3H, CH a), 1.49
3
3
(s, 3H, CH b), 2.37 - 2.52 (m, 1H, 3-Ha), 3.52 (dd, J = 9.2, 7.5 Hz, 1H, 5-H), 3.69 (dd, J =
3
1
1.3, 8.6 Hz, 1H, 4-Hb), 3.78 (dd, J = 9.4, 6.7 Hz, 1H, 5-H), 4.01 (dd, J = 8.4, 7.2 Hz, 1H, 4-
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Ha), 4.46 - 4.60 (m, 2H, CH Ph), 4.65 (t, J = 4.1 Hz, 1H, 2-Ha), 5.83 (d, J = 3.8 Hz, 1H, 1-
2
Ha), 7.24 - 7.42 (m, 5H, CH Ph).
2
1,2-Di-O-acetyl-3-deoxy-5-(O-benzyl)-α/β-D-apio-L-furanose (19): A solution of 18 (750
mg, 2.84 mmol) in 80% aq. acetic acid (10 mL) was stirred at 80 °C for 8h. The reaction
mixture was evaporated to give the crude intermediate as a syrup. This syrup was dissolved in
pyridine (15 mL) and treated with DMAP (50 mg) and acetic anhydride (2.0 mL, 21.2 mmol).
The solution was stirred at room temperature for 4h. The solvent was removed under vacuum
and the resulting residue was purified by silica-gel column chromatography (20% EtOAc-
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
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0
hexanes) to yield 19 (500 mg, 57%) as a colorless oil (α:β anomeric ratio 1:4). Major isomer
1
H NMR (300 MHz, CDCl ) δ ppm 1.94 (s, 3H, Ac), 1.97 (s, 3H, Ac), 2.83 - 2.96 (m, 1H, 3-
3
H), 3.40 (dd, J = 9.1, 7.3 Hz, 1H, 5-H), 3.55 (dd, J = 9.1, 7.6 Hz, 1H, 5-H), 3.77 (t, J = 8.8
Hz, 1H, 4-H), 4.17 (t, J = 8.4 Hz, 1H, 4-H), 4.35 - 4.48 (m, 2H, CH Ph), 5.20 (d, J = 5.0 Hz,
2
13
1
H, 2-H), 6.02 (s, 1H, 1-H), 7.18 - 7.32 (m, 5H, CH Ph). C NMR (75 MHz, CDCl ) δ ppm
2 3
20.5 (CH CO), 21.0 (CH CO), 40.1 (3-C), 66.1 (5-C), 70.9 (4-C), 73.3 (CH Ph), 76.1 (2-C),
3
3
2
9
1
3
9.7 (1-C), 127.6 (C Ph), 127.7 (C Ph), 128.4 (C Ph), 137.8 (C Ph), 169.4 (CH CO),
o p m ipso 3
+
69.7 (CH CO). HRMS (ESI-TOF) m/z: [M + Na] Calcd for C H O Na 331.1158; Found
3
16 20
6
31.1152.
[18]
1
-O-Methyl-2-O-acetyl-3-deoxy-5-(O-benzyl)-β-D-apio-L-furanose (20) : A solution of
18 (2.26 g, 8.55 mmol) and p-TsOH (700 mg, 4.06 mmol) in MeOH (60 mL) was stirred at
room temperature for 16h, neutralized with TEA and evaporated. The residue was partitioned
between EtOAc and water, organic layer separated, dried over anhydrous MgSO and
4
evaporated. The residue was purified by column chromatography (20-40% EtOAc-hexanes).
The intermediate was dissolved in pyridine (15 mL), acetic anhydride (2.4 mL, 25.2 mmol)
and DMAP (200 mg, 1.68 mmol) were added at 0 °C and the reaction mixture was stirred at
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room temperature for 4h. The reaction mixture was evaporated, and partitioned between
EtOAc and 10% aq. KHSO . The organic layer was dried over anhydrous MgSO , filtered
4
4
and evaporated. The residue was purified by silica gel column chromatography (15% EtOAc-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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hexanes) to give 20 (1.85 g, 77%) as a colorless oil. H NMR (300 MHz, CDCl ) δ ppm 2.00
3
(s, 3H, 2-OAc), 2.88-3.02 (m, 1H, 3-H), 3.34 (s, 3H, 1-OMe), 3.46 (dd, J = 9.1, 7.3 Hz, 1H,
5
1
-H), 3.62 (dd, J = 9.2, 7.2 Hz, 1H, 5-H), 3.78 (t, J = 8.6 Hz, 1H, 4-H), 4.14 (t, J = 8.5 Hz,
H, 4-H), 4.46 (d, J = 12.0 Hz, 1H, CH Ph), 4.52 (d, J = 12.0 Hz, 1H, CH Ph), 4.83 (s, 1H, 1-
2
2
H), 5.16 (d, J = 5.3 Hz, 1H, 2-H), 7.27 - 7.39 (m, 5H, CH Ph).
2
[18]
1
,2-O-Isopropylidene-β-D-apio-L-furanose (22) : Compound 21 (5.0g, 21.72 mmol) was
dissolved in 50 mL of 2:1 acetic acid-water mixture and stirred at room temperature for 3
days. Solvents were evaporated in vacuo and silica gel column chromatography of the residue
1
(
50% EtOAc-hexanes) afforded the title compound 22 as a white solid (3.4 g, 83%). H NMR
(300 MHz, CDCl ) δ ppm 1.33 (s, 3H, C(CH ) ), 1.51 (s, 3H, C(CH ) ), 2.12 (t, J = 5.9 Hz,
3
3 2
3 2
1
5
H, 5-OH), 2.69 (s, 1H, 3-OH), 3.71 (dd, J = 6.3, 11.16 Hz, 1H, 4-H), 3.80 (d, J = 9.8 Hz, 1H,
-H), 3.94 (d, J = 9.4 Hz, 1H, 5-H), 3.96 (dd, J = 5.4, 7.5 Hz, 1H, 4-H), 4.38 (d, J = 3.8 Hz,
1H, 2-H), 5.99 (d, J = 3.7 Hz, 1H, 1-H).
[18,20]
1
,2-O-Isopropylidene-5-(O-benzyl)-β-D-apio-L-furanose (23)
: Compound 22 (3.1 g,
1
6.3 mmol) and dibutyltin oxide (6.7 g, 26.9 mmol) was dissolved in toluene (120 mL)
refluxed at 140 °C for 2h. The reaction mixture was allowed to attain 100 °C then added
tetrabutylammonium bromide (2.63 g, 8.15 mmol) and benzyl bromide (3.0 mL, 25.26
mmol). The reaction mixture was stirred at this temperature for 18h. Solvent was evaporated
under reduced pressure and the residue purified by silica gel column chromatography (30%
1
EtOAc-hexanes) to afford 23 (4.3 g, 94%) as a white solid. H NMR (300 MHz, CDCl ) δ
3
ppm 1.32 (s, 3H, C(CH ) a), 1.48 (s, 3H, C(CH ) b), 2.76 (d, J = 0.9 Hz, 1H, 3-OHa), 3.54 (d,
3
2
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J = 9.7 Hz, 1H, 5-H), 3.80 (d, J = 9.7 Hz, 1H, 5-H), 3.82 (dd, J = 9.4, 0.9 Hz, 1H, 4-Ha), 3.88
(dd, J = 9.4 Hz, 1H, 4-Hb), 4.35 (dd, J = 3.5, 0.9 Hz, 1H, 2-Ha), 4.57 (d, J = 12.0 Hz, 1H,
PhCH ), 4.64 (d, J = 12.0 Hz, 1H, PhCH ), 5.98 (d, J = 3.5 Hz, 1H, 1-Ha), 7.27 - 7.42 (m, 5H,
2
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PhCH2).
1,2,3-Tri-(O-acetyl)-5-(O-benzyl)-β-D-apio-L-furanose (24): Following the procedure
described for the synthesis of 17, (2.5g, 8.92 mmol) of 23 rendered pure product 24 (2.45 g,
1
7
5%) as an oil. H NMR (300 MHz, CDCl ) δ ppm 1.96 (s, 3H, COCH ), 2.07 (s, 3H,
3
3
COCH ), 2.10 (s, 3H, COCH ), 3.89 (d, J = 10.5 Hz, 1H, 4-H), 4.05 (d, J = 10.3 Hz, 1H, 4-H),
3
3
4
5
.22 (d, J = 10.3 Hz, 1H, 5-H), 4.34 (d, J = 10.3 Hz, 1H, 5-H), 4.50 - 4.62 (m, 2H, PhCH2),
.49 (d, J = 1.2 Hz, 1H, 2-H), 6.08 (d, J = 1.2 Hz, 1H, 1-H), 7.26 - 7.40 (m, 5H, PhCH2).
[
18]
1
,2-O-Isopropylidene-3-deoxy-β-D-apio-L-furanose (25) : Compound 18 (3.7 g, 14
mmol) was dissolved in methanol (100 mL), to this was added Pd-C (3.7 g, 10% Pd, wet
~50% H O). Stream of hydrogen gas was bubbled through the reaction mixture for 5h at
2
room temperature. The catalyst was filtered off and the filtrate concentrated to give crude
product which on purification by silica-gel column chromatography (40% EtOAc-hexanes)
1
rendered 25 (2.2 g, 90%) as a colorless oil. H NMR (300 MHz, CDCl ) δ ppm 1.34 (d, J =
3
0
5
.6 Hz, 3H, C(CH ) ), 1.53 (s, 3H, C(CH ) ), 2.21 (br.s, 1H, 5-OH), 2.34 (ddtd, J = 11.4, 6.9,
3 2 3 2
.97, 6.0, 4.8 Hz, 1H, 3-H), 3.82 - 3.91 (m, 3H, 4-H & 5-H’s), 3.97 (dd, J = 8.5, 7.3 Hz, 1H,
4-H), 4.73 (t, J = 4.4 Hz, 1H, 2-H), 5.86 (d, J = 3.8 Hz, 1H, 1-H).
1
,2-O-Isopropylidene-3-deoxy-α-D-apio-D-furanose (26): To a solution of compound 25
(2.2 g, 12.63 mmol) in 400 mL of acetone was added concentrated sulfuric acid (2.2 mL) and
the mixture was stirred at room temperature for 1.5h. Then sodium carbonate (14 g) was
added and stirred at room temperature for 45 minutes. Inorganic salts were removed by
filtration and the filtrate concentrated under reduced pressure to afford oil. TLC indicated the
2
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conversion in favor of required isomer (roughly 2:1). The title compound is slightly polar
with respect to starting material (R after two runs: 0.35 for 26 and 0.4 for 25; eluent, 2.5%
f
MeOH in CH Cl ). Silica-gel flash column chromatography (0.5-1.5% MeOH in CH Cl )
2
2
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
afforded title compound and starting material. After three cycles 1.6 g (73%) of 26 was
1
procured as a colorless oil. H NMR (300 MHz, CDCl ) δ ppm 1.31 (d, J = 0.6 Hz, 3H,
3
C(CH )b), 1.50 (s, 3H, C(CH )a), 1.89 (br.s, 1H, 5-OH), 2.36 - 2.46 (m, 1H, 3-H), 3.58 (dd, J
3
3
=
6.6, 3.4 Hz, 2H, 5-CH ), 3.83 (d, J = 9.1 Hz, 1H, 4-Hb), 4.10 (dd, J = 8.9, 5.1 Hz, 1H, 4-
2
1
3
Ha), 4.60 (d, J = 3.5 Hz, 1H, 2-H), 5.81 (d, J = 3.8 Hz, 1H, 1-H). C NMR (75 MHz, CDCl )
3
δ ppm 26.2 (C(CH )b), 26.8 (C(CH )a), 48.1 (3-C), 62.0 (5-C), 68.7 (4-C), 82.3 (2-C), 105.6
3
3
(
1-C), 111.3 (C(CH3)2).
1
,2-O-Isopropylidene-3-deoxy-5-(O-benzyl)-α-D-apio-D-furanose (27): To an ice cold
solution of 26 (1.6 g, 9.2 mmol) in DMF (30 mL) was added NaH (60% in mineral oil, 0.55g,
13.8 mmol) and then benzyl bromide (1.64 mL, 13.8 mmol) dropwise. The reaction mixture
was stirred at room temperature overnight. Methanol (5 mL) was added and stirred for further
3
0 minutes. The volatile materials were removed under vacuo and the residue was partitioned
between ethyl acetate and water. The organic layer was separated, dried over anhydrous
Na SO , evaporated and the residue purified by column chromatography (5-15% EtOAc in
2
4
1
hexanes) to afford 27 (2.3 g, 95%) as a colorless oil. H NMR (300 MHz, CDCl ) δ ppm 1.31
3
(d, J = 0.6 Hz, 3H, C(CH )b), 1.51 (s, 3H, C(CH )a), 2.56 (td, J = 7.5, 5.1 Hz, 1H, 3-Ha),
3
3
3
4
1
2
.37 (d, J = 7.6 Hz, 2H, 5-CH ), 3.83 (d, J = 8.8 Hz, 1H, 4-Hb) 4.09 (dd, J = 8.9, 5.1 Hz, 1H,
2
-Ha) 4.51 (d, J = 3.2 Hz, 2H, PhCH ), 4.56 (d, J = 3.5 Hz, 1H, 2-Hb), 5.79 (d, J = 3.5 Hz,
2
1
3
H, 1-Hb), 7.27 - 7.41 (m, 5H, PhCH ). C NMR (75 MHz, CDCl ) δ ppm 26.3 (C(CH )b),
2
3
3
6.9 (C(CH )a), 46.1 (3-C), 68.8 (4&5-C), 73.3 (PhCH ), 82.4 (2-C), 105.6 (1-C), 111.2
3
2
+
(C(CH ) ) 127.8, 127.9, 128.6, 138.1 (PhCH ). MS (ESI-TOF) m/z: [M + K] Calcd for
3
2
2
C H O K 303.0999; Found 303.1078.
15
20
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