Scaleable processes for the synthesis of (-)-β-d-2,6-diaminopurine dioxolane (Amdoxovir, DAPD) and (-)-β-d-2-aminopurine dioxolane (APD)

Article abstract of DOI:10.1016/j.tet.2012.05.039

An efficient and scalable synthesis of (-)-DAPD and (-)-APD has been developed. We discovered that t-butyl cyanoacetate can be used as a new additive for the sugar nucleoside base coupling step en route to DAPD with improved β-selectivity and an isolated yield four-fold greater than the original process scale method. Using this new process, (-)-DAPD has been prepared on greater than 20 g scale. In the synthesis of (-)-APD, a key enzyme-catalyzed hydrolysis reaction afforded the water soluble deprotected α-anomer while leaving the β-anomer completely untouched.

Full text of DOI:10.1016/j.tet.2012.05.039

Tetrahedron 68 (2012) 5738e5743
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Scaleable processes for the synthesis of (ꢀ)-
b- -2,6-diaminopurine dioxolane
-2-aminopurine dioxolane (APD)
D
(Amdoxovir, DAPD) and (ꢀ)-
b
-D
Longhu Zhou a, Steven J. Coats b, Hongwang Zhang a, Junxing Shi b, Drew R. Bobeck b,
,
Raymond F. Schinazi a
*
a Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center,
Atlanta GA 30033, USA
b RFS Pharma LLC, 1860 Montreal Road, Tucker, GA 30084, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and scalable synthesis of (ꢀ)-DAPD and (ꢀ)-APD has been developed. We discovered that t-
Received 28 February 2012
Received in revised form 1 May 2012
Accepted 9 May 2012
butyl cyanoacetate can be used as a new additive for the sugar nucleoside base coupling step en route to
DAPD with improved
b-selectivity and an isolated yield four-fold greater than the original process scale
method. Using this new process, (ꢀ)-DAPD has been prepared on greater than 20 g scale. In the synthesis
Available online 16 May 2012
of (ꢀ)-APD, a key enzyme-catalyzed hydrolysis reaction afforded the water soluble deprotected
a-
anomer while leaving the
b
-anomer completely untouched.
Keywords:
Amdoxovir
DAPD
Published by Elsevier Ltd.
Diaminopurine
Dioxolane
Nucleoside
Glycosylation
NH2
1. Introduction
N
N
N
N
N
HO
N
HO
The World Health Organization (WHO) reported that there were
more than 34 million individuals living with HIV and about 2 million
deaths from AIDS worldwide in 2010, including 250,000 children.1
During the last two decades, many drugs and treatment regimes for
N
NH2
N
NH2
O
O
O
O
(-)-DAPD
(-)-APD
-D-2-aminopurine di-
Fig. 1. (ꢀ)-
b
-D-2,6-Diaminopurine dioxolane (DAPD) and (ꢀ)-
b
this disease have been successfully discovered and developed. (ꢀ)-
b-
oxolane (APD).
D-2,6-Diaminopurine dioxolane (Amdoxovir, DAPD), a prodrug of
(ꢀ)-
b-D-dioxolane guanine (DXG), was found to exhibit good anti-
HIV-1 activity in both humans2 and against virus containing the
M184 V/I mutation,3,4,10a and is currently in advanced Phase 2 clinical
Since the first racemic dioxolane nucleosides were reported by
Belleau8 in 1989, a number of enantiomerically pure dioxolane
analogs have been synthesized and identified as potent and non-
toxic anti-HIV agents. For example, the Chu/Schinazi groups9 and
later Gu et al.3a,10a reported (ꢁ)-DAPD and (ꢁ)-DXG both inhibited
HIV-l in various cell systems. A process for obtaining enantio-
trials5 (Fig.1). (ꢀ)-
b-D-2-Aminopurine dioxolane (APD), a more water
soluble prodrug of DXG, also exhibits excellent anti-HIV activity in
animal models as well as an enhanced pharmacokinetic profile in
rhesus monkeys.6,7 Herein, wedescribeimprovedsyntheticroutes for
these important anti-HIV nucleoside analogs. For the synthesis of
merically pure
synthesis has also been reported (Scheme 1). The fourteen-step
-mannose11 resulted in
a modest overall yield. It involved a difficult and low yielding
Pb(OAc)4 oxidation to dioxolane acetate, 112 and multiple chroma-
tography purification steps that make the process unsuitable for
commercial development. It is noteworthy that reaction of in-
termediate 2 with ammonia gave a mixture of both 6-Cl and 2-F
substitution with 2-flouro ipso displacement representing the
b-D-1,3-dioxolane nucleosides via a stereospecific
€
DAPD, a cyanoacetate was employed as an additive in the Vobruggen
glycosylation reaction to improve the
b
b/a anomeric ratio and overall
anomer isolated yield. A scale-limiting chromatographic purifica-
tion was eliminated through the use of an enzyme-catalyzed hy-
synthesis of (ꢀ)-DAPD from 1,6-anhydro-
D
drolysis in the synthesis (ꢀ)-APD.
* Corresponding author. E-mail address: rschina@emory.edu (R.F. Schinazi).
0040-4020/$ e see front matter Published by Elsevier Ltd.
doi:10.1016/j.tet.2012.05.039

L. Zhou et al. / Tetrahedron 68 (2012) 5738e5743
5739
major product occurring in the presence of the fairly reactive 6-Cl
substituent.
with sodium azide in DMF, filtration, and direct palladium cata-
lyzed hydrogenation. Excess NaN3 was consumed prior to filtration
by the addition of n-butyl bromide. Removal of the isobutyl ester
was effected with butylamine in refluxing methanol to reveal DAPD
that was crystallized in denatured ethanol:water to provide active
pharmaceutical ingredient (API) quality (ꢀ)-DAPD. The large scale
use of sodium azide provided an additional challenge in commer-
cial development (Scheme 2). Despite these shortcomings, over
100 kg of (ꢀ)-DAPD has been prepared utilizing this process.
More recently the glycosylation of 2,6-diaminopurines or silyl
2,6-diaminopurines with dioxolane acetates was reported to be
facilitated by the presence of a 1,3-dicarbonyl compound, such as t-
butyl acetoacetate.16 Although it is an improvement, this process
O
HO
O
OAc
9 steps
O
OH OH
OH
O
1
1,6-anhydro-D-mannose
Cl
N
NH2
N
N
N
N
t-Bu(Ph)2SiO
HO
N
N
F
N
NH2
O
O
O
O
suffers in that only a 33% yield of the desired
from the critical glycosylation step (Table 1, entry 2). Interestingly,
the ratio was 1:1.8 whereas in the absence of t-butyl acetoa-
cetate the ratio was 1:1.2 (Table 1, entry 1) close to the roughly
b-isomer is obtained
(-)-DAPD
2
Scheme 1. Early synthesis of (ꢀ)-DAPD.
a/b
a/b
The next significant advancement for the dioxolane nucleosides
was the development of a more efficient synthesis of the dioxolane
nucleoside analogs that included an enzyme driven resolution of
the R/S enantiomers as well as a more scalable overall process.13
The resolution relies on a simple two-step synthesis of racemic,
ester protected, dioxolane lactone from commercially available 2,2-
dimethoxy or 2,2-diethoxy ethanol in an isolated 79% yield. An
enzyme resolution of the racemic dioxolane lactone completes the
three-step sequence to the key intermediate (R)-lactone B, 3
(Scheme 2).14 The new synthesis was scaled to produce over one
thousand kilograms of (R)-lactone B, 3.15
equal mix of anomers that had been observed with all previous
dioxolane glycosylation reactions. The role played by the glycosyl-
ation additive t-butyl acetoacetate was not discussed by the au-
thors and our studies in this regard provided no clear explanation
for the improved yield and
b-selectivity. To support our clinical
efforts, it is advantageous for us to continue the development of
a more cost-effective process scalable synthesis for preparing API
quality dioxolane nucleoside derivatives, such as (ꢀ)-DAPD and
(ꢀ)-APD.
Table 1
Glycosylationa results from the reaction of 2,6-diaminopurine 9 with dioxolane
acetate 4
RO
O
a
b
OAc
O
O
Entry
Additive
Solvent
Lewis acid
Yield(%)
b/a
O
N
O
O
O
1
2
3
4
5
6
7
8
None
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH3CN
CHCl3
TMSI
TMSI
TMSI
TMSI
TMSI
TMSI
SnCl4
41
62b
53
45
72c
54d
0
34
11
14
55
51
50f
1.2
1.8
1.3
1.7
2.2
2.4
NA
0.8
0.8
0.8
1.5
1.8
2.6
CH3COCH2COOtBu
NCCH2COOMe
NCCH2COOEt
NCCH2COOtBu
NCCH2COtBu
NCCH2COOtBu
NCCH2COOtBu
None
R = (CH3)2CHCO
4
(R)-lactone B, 3
N3
Cl
N
N
N
N
d
c
N
TiCl3(OiPr)
TMSOTf
TMSOTf
TMSI
TMSI
TMSI
RO
RO
N
N
N3
Cl
O
O
9
10
11
12
13
NCCH2COOtBu
NCCH2COOtBu
NCCH2COOtBu
NCCHC(OTMS)tBue
O
O
6
CH2Cl2
NH2
N
NH2
N
a
Conditions for the glycosylation reaction: (i) HMDS, (NH4)2SO4, reflux, (ii) ad-
N
N
N
N
ditive, Lewis acid, ꢀ10eꢀ15 ꢂC; 1 h; 4 ꢂC, 15 h, rt for 3 h.
e
b
RO
HO
33% Isolated yield of
45% Isolated yield of
33% Isolated yield of
b
b
b
-isomer.
-isomer.
-isomer.
N
NH2
N
NH2
c
d
e
f
O
O
O
O
98:2 Z:E-enol silane based on 1H NMR.
(-)-DAPD
Total yield: 9-18%
7
31% Isolated yield of b-isomer.
Scheme 2. Reagents and conditions: (a) (i) LiAl(Ot-Bu)3H, THF, ꢀ20 ꢂC, 2 h; (ii) Ac2O,
DMAP, ꢀ10 ꢂC, 2 h, rt, overnight, 60%; (b) (i) 2,6-dichloropurine, 5, HMDS, (NH4)2SO4,
reflux, 2 h; (ii) TMSI, CH2Cl2, ꢀ10 ꢂC to rt, 6 h, 9e18%; (c) (i) NaN3, DMF, rt, 1 h; (ii) n-
BuBr, rt, 0.5 h; (d) H2, 5% Pd/C, DMF, rt; (e) (i) n-butyl amine, MeOH, reflux, 4 h; (ii)
recrystallization denatured ethanol:water 52:48 w/w 61e91% (four steps).
2. Results and discussion
2.1. Synthesis of (L)-DAPD
This more efficient dioxolane nucleosides synthesis was used to
prepare kilogram quantities for the first human clinical trials.13 As
shown in Scheme 2, this process started with the selective re-
duction of (R)-lactone B, 3 with lithium tri-tert-butoxyaluminum
hydride followed by acylation with acetic anhydride to provide (2R-
4R/S)-4-acetoxy-2-isobutyryloxymethyl-1,3-dioxolane, 4. In the
key coupling step, 2,6-dichloropurine, 5 and dioxolane acetate, 4
Herein we report a more robust process for the large scale
synthesis of (ꢀ)-DAPD starting from (R)-lactone B, 3. Dioxolane
acetate, 4 was prepared from 3 as described in Scheme 2. Although
our studies to uncover the role of t-butyl acetoacetate in the higher
yielding and more facial selective dioxolane glycosylation were
inconclusive we envisioned an active methylene derived complex
€
were used under Vobruggen conditions to afforded a crude mixture
or adduct that preferentially blocks the
a-face leading to a prefer-
of nucleoside analogs as a 1:1 mixture of
a- and b-anomers. A single
ence for the incoming nucleoside base to attack the
b
-face of the
recrystallization provided the pure -anomer, 6. Unfortunately, the
b
dioxolane oxonium complex. The overall yield and anomeric ratios
yield ranged from a disappointing 9e18%. A two-step conversion to
the desired 2,6-diaminopurine nucleoside involved treatment of 6
with and without t-butyl acetoacetate are shown in Table 1 entries
1 and 2. Part of our study involved investigation of
a-cyano

5740
L. Zhou et al. / Tetrahedron 68 (2012) 5738e5743
NH2
N
carbonyl compounds as additives along with variation of the sol-
vent and Lewis acid in the glycosylation reaction of dioxolane ac-
etate, 4 with silylated 2,6-diaminopurine (Table 1). We found that
methyl cyanoacetate (Table 1, entry 3) had very little effect on the
yield or anomeric ratio versus the reaction without additives (Table
N
N
a
4
RO
N
NH2
NHTMS
N
O
N
O
8, β-isomer: 45%
N
TMS
TMSHN
N
1, entry 4). Ethyl cyanoacetate gave a 1:1.7 ratio of
a- and b-
R = (CH3)2CHCO
anomers favoring the -anomer, however, in an unimproved overall
b
yield of 45% (Table 1, entry 4). When the dioxolane acetate, 4 was
coupled with silylated 2,6-diaminopurine in the presence of the
c
b
NH2
additive t-butyl cyanoacetate (Table 1, entry 5) a 1:2.2 ratio of
and -anomers in a much improved 72% total yield was obtained
and the -isomer 8 was obtained in an isolated 45% yield after
a single recrystallization from isopropanol. The corresponding t-
butyl cyanoketone (Table 1, entry 6) gave a similar yield of 70% but
a-
N
N
NH2
b
N
HO
N
N
N
NH2
b
O
N
H
H2N
N
O
(-)- DAPD
9
the 1.5:1 b/a anomeric ratio was much less favorable when com-
Yield: 22% from 4
pared to t-butyl cyanoacetate.
Scheme 3. Reagents and conditions: (a) CNCH2COOtBu, TMSI, CH2Cl2, ꢀ10eꢀ15 ꢂC; 1 h;
4 ꢂC, 15 h, rt for 3 h, 45%; (b) HMDS, (NH4)2SO4, reflux, 4 h; (c) NH3/MeOH, rt, 25 h, 83%.
With t-butyl cyanoacetate identified as a superior additive var-
ious Lewis acids and solvents were evaluated to further optimize
the dioxolane glycosylation conditions. Although tin (IV) chloride
was employed with good results as a Lewis acid in the dioxolane
and oxathiolane pyrimidine nucleoside synthesis,17 no desired
product was detected in the synthesis of DAPD from 2,6-
diaminopurine (Table 1, entry 7). When TiCl3(OiPr)7 was used
only low yields and poor b-isomer selectivity was observed (Table 1,
entry 8). Trimethylsilyl trifluorosulfonate (TMSOTf) both with and
without t-butyl cyanoacetate gave only low yields, which surpris-
hydroxide/methanol provided simultaneous reduction and depro-
tection to provide the desired (ꢀ)-APD nucleoside (Scheme 4).
Cl
N
N
a
b
4
RO
N
N
NH2
O
ingly favored the
a-anomer (Table 1, entries 9 and 10). Iodo-
O
trimethylsilane (TMSI) was found to be the best Lewis acid tested
and it was used to test a few of the common glycosylaton solvents.
Both acetonitrile and chloroform proved inferior to dichloro-
methane in both yield and anomer selectivity (Table 1, entries 11
and 12). In an effort to gain mechanistic insight into these glyco-
sylation conditions we attempted to prepare the TMS ketene acetal
of t-butyl cyanoacetate for use as an alternate additive. Despite the
use of hexamethyldisilazane, TMSCl, and TMSOTf as silylation
agents with either triethylamine or lithium diisopropyl amide as
base, we were unable to isolate or detect the presence of TMS ke-
tene acetal by 1H NMR. Conversely the t-butyl cyanoketone was
readily converted to the TMS enol ether by exposure to LDA/TMSCl
and its use as an additive in the glycosylation reaction gave a 2.5:1
10
Cl
N
N
N
N
N
N
c
HO
N
NH2
RO
N
NH2
O
O
O
O
11
R = (CH3)2CHCO
(-)-APD
Yield: 23% from 4
Scheme 4. Reagents and conditions: (a) TMSI, CHCl3, silylated 2-amino-6-chloropurine
ꢀ10eꢀ15 ꢂC, 2 h; rt, 15 h; reflux, 5 h; (b) silica gel chromatography (hexane:ethyl
acetate 100:0 to 0:100) 46% from 4; (c) H2/PdeC, NH4OH/MeOH, rt, 20 h, 84%.
In this sequence, the key difficulty for kilogram scale synthesis
b
/
a
anomeric ratio; the highest ratio observed under any conditions
(Table 1, entry 13). Unfortunately the isolated yield of the pure
anomer was less than half that observed under the best condi-
tions. By using the additive t-butyl cyanoacetate and dichloro-
methane as solvent the ratio of was improved and the isolated
yield of -isomer, 8, obtained from the anomer mixture by a single
of (ꢀ)-APD was the separation of pure
b-isomer, 11 from the b/
mixture, 10 where on small scale flash chromatography was
a
b
successfully employed (Scheme 4). As the reaction was scaled to
larger amounts crystallization from a large number of solvent
systems was investigated but was not successful at separating the
b/a
b
desired b-isomer, 11.
crystallization, was increased to 45%. This represents a 37% im-
provement in the isolated yield of 8 versus that obtained with t-
butyl acetoacetate. (ꢀ)-DAPD was obtained in high yield after
deprotection using methanol saturated with ammonia to provide
an overall 22% yield of (ꢀ)-DAPD based on the starting (R)-lactone
B, 3 (Scheme 3).
Although enzymes have been employed widely for biocatalytic
resolutions,18 little information exists regarding the enantiose-
lective enzyme-catalyzed hydrolysis of racemic nucleosides.19
Wengel and co-workers20 reported the first use of esterase cata-
lyzed diastereoselective deacetylation to solve the problem of nu-
cleoside anomer separation. Thus, a lipase-catalyzed deacylation of
an anomeric mixture of peracetylated 20-deoxyribofuranosyl thy-
mines was carried out using two enzymes: wheat germ lipase
2.2. Synthesis of (L)-APD
(WGL) and porcine liver esterase (PLE). The b-anomer of thymidine
An optimized TMSI mediated glycosylation reaction has been
previously reported for the synthesis of APD.7 In an effort to further
improve this reaction, we applied the above optimized conditions
to the synthesis of (ꢀ)-APD, and surprisingly, the additives t-butyl
cyanoacetate and t-butyl acetoacetate had no effect on the outcome
of the anomeric ratio in the glycosylation reaction. This implicates
the involvement of the purine base in the facially selective glyco-
sylation step. Our studies also revealed that 6-chloro-2-
aminopurine consistently gave significantly better yields of the
desired beta isomer when compared to 2-aminopurine. A simple
high yielding catalytic hydrogenation of 11 in ammonium
was the only completely deprotected nucleoside product.
In order to realize a more scaleable process for the synthesis of
(ꢀ)-APD we examined the potential for selective enzymatic de-
esterification of the
10. If successful, the resulting alcohol containing
a
-anomer of the
b
/
a
mixture of nucleosides,
-anomer, 13,
would be significantly more polar thus facilitating its separation
from the intact -anomer, 11. Pseudomonas fluorescens lipase (PFL),
a
b
porcine liver esterase (PLE) and Candida antarctica lipase (CAL)
were chosen because of their commercial availability, relative low
cost, and tolerance for a wide class of substrates. The hydrolysis

L. Zhou et al. / Tetrahedron 68 (2012) 5738e5743
5741
reactions were typically incubated at room temperature in 1 mL of
4. Experimental
4.1. General
pH 7.0 buffer (0.05 M potassium phosphate) and 1 mL of organic
solvent with 50 mg of nucleoside 10 and enzymes PFL, CAL or PLE
(25 mg, 25 mg or 100 units, respectively). The hydrolysis reactions
were monitored by thin layer chromatography and high pressure
liquid chromatography-mass spectrometry (LC/MS). The results are
summarized in Table 2. When PFL was employed no hydrolysis of
10 took place in tetrahydrofuran (THF), methanol (MeOH), or di-
methyl formamide (DMF) (entries 1e3, Table 2), and only trace
Anhydrous solvents (DrisolvÒ) were purchased from EMD
Chemicals Inc (Philadelphia, PA). Reagents were purchased from
commercial sources. Unless noted otherwise, the materials used in
the examples were obtained from readily available commercial
suppliers or synthesized by standard methods known to one skilled
in the art of chemical synthesis. Thin layer chromatography (TLC)
was carried out on precoated silica gel thin layer sheets 60 F254
from EMD (E. Merck, Darmstadt, Germany) or on Silica Gel Uniplate
amounts of the a-isomer was hydrolyzed with CH3CN as co-solvent
(entry 4, Table 2). No reaction was observed when PLE was used in
THF, MeOH, or DMF (entries 5e7, Table 2). When the PLE reaction
was conducted in CH3CN, the starting material 10 was completely
but nonselectively converted to nucleosides 12 and 13. Fortunately,
selective enzyme-catalyzed hydrolysis took place when CAL was
GHLF 250 m
m (Analtech, Newark DE, part number 21521). 1H NMR
spectra were taken on a Varian Unity Plus 400 spectrometer (Var-
ian, Inc., Polo Alto, CA, USA) at room temperature and reported in
parts per million downfield from internal tetramethylsilane. Signal
multiplicities are represented by s (singlet), d (doublet), dd (dou-
blet of doublets), t (triplet), q (quadruplet), br (broad), bs (broad
singlet), m (multiplet). All J values are in hertz. Mass spectral
analyses were performed on a Micromass TOF instrument (Hew-
lettePackard HPLC driven electrospray MS instrument). Analytical
HPLC analyses were performed on a HewlettePackard HPLC with
used with all four solvents (entries 9e12, Table 2). The
10 can be selectively hydrolyzed leaving the -isomer intact. Re-
covery of the desired unreacted -anomer, ester 10, was accom-
plished by a simple extraction, which left the undesired -anomer
in the water layer. The -anamer 10 was obtained sufficiently pure
a-isomer of
b
b
a
b
to use in further reactions without additional purification. Simul-
taneous removal of the isobutyl ester and reduction of the 6-chloro
group was performed by hydrogenation with Pd/C as catalyst in
MeOH containing NH4OH. High purity (ꢀ)-APD was obtained in
a highly scalable process by filtering the resulting material through
a short silica gel column followed by crystallization in CH2Cl2/
MeOH. The overall yield of APD from (R)-lactone B was 23%
(Scheme 4).
a Phenomenex Gemini-NX column (2 mmꢃ50 mm, 3
mm, C18,
ꢀ
110 A). Mobile phase flow was 0.7 ml/min with a 3.5 min gradient
from 96% aqueous media (0.05% formic acid) to 96% CH3CN (0.05%
formic acid) with a 5.5 min total acquisition time and 190e360 nm
PDA detection.
4.1.1. Silylation of 2,6-diaminopurine. 2,6-Diaminopurine,
9
(750 mg, 5.0 mmol), ammonium sulfate (750 mg, 5.7 mmol), and
hexamethyldisilazane (20 mL) were added to a 250 mL three-neck
flask. The suspension was heated to reflux with stirring at
130e135 ꢂC (oil-bath) for 4 h. During this period the solution be-
came homogeneous. The solution was cooled to 85 ꢂC and the ex-
cess hexamethyldisilazane was subsequently removed by
distillation at reduced pressure. After the hexamethyldisilazane
was completely removed, the residue was cooled to rt under vac-
uum then 10 mL of anhydrous methylene chloride was added to
prepare a solution of crude silylated 2,6-diaminopurine of suitable
purity for the preparation of dioxolane nucleosides 8. This material
is highly moisture sensitive and therefore used directly in the next
step.
Table 2
Enzyme-catalyzed reactions of nucleosides 10
HO
Cl
O
N
O
N
N
NH2
Enzyme, Solvent
N
N
10
+
N
HO
N
NH2
pH 7.0 potassium
phosphate buffer
N
O
O
Cl
12
13
undesired
desired
Entry
Enzyme
Solvent
Reaction time (h)
Conversion (%)
12 13
1
2
3
4
5
6
7
8
PFL
PFL
PFL
PFL
PLE
PLE
PLE
PLE
CAL
CAL
CAL
CAL
THF
MeOH
DMF
CH3CN
THF
MeOH
DMF
CH3CN
THF
MeOH
DMF
24
24
24
24
24
30
24
15
15
18
18
24
0
0
0
0
0
0
0
0
0
0
4.1.2. ((2R)-4-Acetoxy-1,3-dioxolan-2-yl)methyl isobutyrate (4). To
a well stirred solution of LiAl(OtBu)3H (25.4 g, 100.0 mmol) in dry
THF (150 mL) at e10 to e20 ꢂC was added a precooled (R)-(4-oxo-
1,3-dioxolan-2-yl)methyl isobutyrate (12.5 g, 66.4 mmol) over
a period of 10 min under N2 atmosphere. The reaction mixture was
allowed to stir for 2 h at e10 to e20 ꢂC. To this solution DMAP (7.0 g,
57 mmol) was added in one portion and stirred for 30 min followed
by dropwise addition of Ac2O (46.0 mL, 443 mmol). After stirring
the bright yellow solution for 2 h at e10 ꢂC, the reaction was
allowed to slowly raise to room temperature and stirred overnight.
The dark brown solution was poured into saturated aqueous NH4Cl
(180 mL), stirred for 30 min, filtered (to remove Li salts), concen-
trated in vacuo and extracted with ethyl acetate (3ꢃ60 mL). The
combined organic solutions were washed with saturated aqueous
NaHCO3 (2ꢃ50 mL), brine (1ꢃ25 mL), dried over anhydrous Na2SO4,
filtered, and evaporated under reduced pressure to afford crude 4
(red syrup). This crude material is suitable for immediate use in
glycosylation reactions. Alternatively, it can be stored for extended
periods at ꢀ80 ꢂC protected from air and moisture as a 1 M solution
in CHCl3. Rf: 0.45 (ethyl acetate:hexanes 1:4). 1H NMR analysis
Trace
0
0
0
100
0
100
100
100
100
100
9
10
11
12
0
0
0
CH3CN
PFL: Pseudomonas fluorescens lipase; PLE: porcine liver esterase; CAL: lipase acrylic
resin from Candida antarctica.
3. Conclusions
A novel, scalable and efficient processes for the synthesis of
(ꢀ)-DAPD and (ꢀ)-APD was developed. We found that t-butyl
cyanoacetate can be used as an additive for the coupling step in
route to (ꢀ)-DAPD and resulted in improved
b-selectivity with an
isolated yield four times greater than the original process scale
method. The mechanistic role of this new additive and its en-
hancement of
b-selectivity are currently under investigation. A
difficult silica gel chromatography step in route to (ꢀ)-APD was
showed a 1:1 mixture of
CDCl3)
: 1.18 (d, 6H, J¼7.2 Hz, 2ꢃCH3); 1.19 (d, 6H, J¼6.8 Hz,
2ꢃCH3); 2.09 (s, 3H, CH3); 2.10 (s, 3H, CH3); 2.58e2.61 (m, 2H,
a- and b
eisomers. 1H NMR (400 MHz,
replaced by an enzyme-catalyzed hydrolysis reaction that allowed
d
the pure b-anomer, 10, to be isolated by a simple water extraction.

5742
L. Zhou et al. / Tetrahedron 68 (2012) 5738e5743
2ꢃCH); 4.20e4.42 (m, 8H, 4ꢃCH2); 5.32 (t, 1H, J¼4.4 Hz, CH); 5.42
(t, 1H, J¼4.4 Hz, CH); 6.35 (d, 1H, J¼4.0 Hz, CH); 6.41 (dd, 1H,
J¼4.0 Hz, J¼1.6 Hz, CH).
resulting in 17.1 g (83%) of (ꢀ)-DAPD as colorless crystals, mp:
236e238 ꢂC (lit.21 mp: 236e237 ꢂC). 1H NMR (400 MHz, DMSO-d6)
d:
3.58 (dd, 2H, J¼6.0 Hz, J¼3.2 Hz); 4.17 (dd, 1H, J¼9.5 Hz, J¼5.5 Hz);
4.42 (dd, 1H, J¼9.5 Hz, J¼1.8 Hz); 5.02 (t, J¼3.2 Hz); 5.18 (t, J¼6.0 Hz);
5.88 (brs, 2H); 6.18 (dd,1H, J¼5.5 Hz, J¼1.8 Hz); 6.79 (brs, 2H); 7.86 (s,
Anhydrous CHCl3 was added to a total volume of 40 mL to
prepare a 1 M solution (based on a 60% yield).
1H). 13C NMR (100 MHz, DMSO-d6)
d: 61.9, 71.0, 79.3, 105.9, 113.3,
4.1.3. (2R,4R)-2-Isobutyryloxymethyl-4-(2,6-diaminopurin-9-yl)-
[1,3]-dioxolane (8). To the solution of silylated 2,6-diaminopurine
in dry methylene chloride from above was added 4 mL of 1 M
((2R, 4R/S)-4-acetoxy-1,3-dioxolan-2-yl)methyl isobutyrate, 4 in
chloroform and 0.75 mL of t-butyl cyanoacetate under a nitrogen
atmosphere. The resulting solution was cooled to between ꢀ10 and
ꢀ15 ꢂC then a solution of iodotrimethylsilane (1.5 mL) in 2 mL of dry
methylene chloride was added dropwise over the course of
2e3 min. The mixture was then stirred between ꢀ10 and ꢀ15 ꢂC for
1 h, 4 ꢂC for 15 h and rt for 3 h. The reaction mixture was added
dropwise to 0.5 M hydrochloric acid (18 mL) at 0 ꢂC. The mixture
was warmed with stirring to 25 ꢂC and stirred for another 20 min.
The phases were separated and the organic phase was back-
extracted once with 0.5 M hydrochloric acid (18 mL). The com-
bined aqueous phases were washed twice with 25 mL of methylene
chloride. Then, after addition of a further 50 mL of methylene
chloride, the pH was adjusted to 9.0 with approximately 40 mL of
10% sodium carbonate solution. The mixture was stirred at 25 ꢂC for
1 h and the phases were separated. The aqueous phase was back-
extracted twice with 30 mL of methylene chloride. The combined
organic phases were washed once with 25 mL of water. The solvent
was removed under vacuum and the resulting residue was passed
through a short flash silica gel column, eluting with 0e15% meth-
anol in methylene chloride to yield 940 mg of yellowish solid. LC/
135.4, 152.0, 156.7, 161.0. Calculated for C9H12N6O3: C, 42.86; H, 4.80;
N, 33.32. Found: C, 42.57; H, 4.69; N, 33.02.
4.1.6. Silylation of 2-amino-6-chloropurine. 2-Amino-6-chloropurine
(0.85 g, 5.0 mmol), ammonium sulfate (0.75 g, 5.7 mmol), and
1,1,1,3,3,3-hexamethyl disilazane (HMDS) (3 mL) were heated to
reflux under a N2 atmosphere. A clear solution was obtained after 2 h
of reflux. The reaction mixture was cooled to rt and used directly in
the next step.
4.1.7. Isobutyric acid-4-(R)-(2-amino-6-choloropurin-9-yl)-[1,3]-di-
oxolan-2-(R)-yl methyl ester (11) via chromatography. Isobutyric
acid-4-acetoxy-[1,3]-dioxolan-2-(R)-yl methyl ester, 4 (3.77 mL of
1 M solution in step 1, 3.77 mmol) was dissolved in CHCl3 (10 mL)
and cooled to ꢀ10 to ꢀ20 ꢂC. Iodotrimethylsilane (0.65 mL,
4.8 mmol) was slowly added by syringe over a period of 1 h and
stirred for 2 h at ꢀ10 to ꢀ20 ꢂC. To this solution was added persi-
lylated 2-amino-6-choloropurine, dissolved in CHCl3 (15 mL) and the
reaction was stirred at ꢀ10 to ꢀ20 ꢂC for 2 h. The reaction mixture
was allowed to warm to room temperature, stirred an additional 2 h
at rt and then heated to gentle reflux for 4 h. The resulting mixture
was cooled to rt, quenched with water, washed with saturated
aqueous NaHCO3 (2ꢃ20 mL), saturated aqueous sodium thiosulfate
(20 mL), water (2ꢃ20 mL), and brine (2ꢃ20 mL). The organic layer
was dried over Na2SO4, filtered and evaporated under reduced
MS analysis showed the isomer ratio (
product was crystallized from isopropanol and 690 mg (45% yield)
of colorless -isomer crystals were obtained. NMR analysis revealed
b/
a
ratio¼2.2:1). The crude
pressure to yield a yellow solid consisting of the
10. The residue was purified by silica gel flash column chromatog-
raphy, eluting with 0e100% ethyl acetate in hexane to yield -isomer,
117 as a white foam (590 mg, 45.8%). 1H NMR (400 MHz, CDCl3)
a/b anomer mixture,
b
one mol equivalent of isopropanol in addition to (2R, 4R)-2-
b
isobutyryloxymethyl-4-(2,6-diaminopurin-9-yl)-1,3-dioxolane,
d:
8.13 1H NMR (400 MHz, DMSO-d6)
d
: 0.95e1.04 (m, 12H, 4ꢃCH3);
1.12 and 1.18 (2d, J¼7.2 Hz, 6H, 2ꢃCH3); 2.60 (d, J¼7.2 Hz 1H, CH);
4.28 (dd, J¼5.2 Hz, J¼10.0 Hz 1H, CH2); 4.37 (m, 2H, CH2); 4.54 (dd,
J¼0.8 Hz, J¼10.0 Hz, 1H, CH2); 5.21 (bs, 2H, NH2); 5.31 (t, J¼3.2, 1H,
CH); 6.37 (dd, J¼1.2 Hz, J¼5.2 Hz, 1H, CH) and 8.12 (s, ArH). 13C NMR
2.44e2.40 (m, 1H, CH); 3.71e3.75 (m, 1H, CH), 4.17e4.52 (m, 5H,
CH, 2ꢃCH2); 5.20 (t, 1H, J¼3.2 Hz, CH); 5.80 (brs, 2H, NH2) 6.17 (dd,
1H, J¼1.6 Hz, J¼5.6 Hz, CH); 6.71 (brs, 2H, NH2); 7.74 (s, 1H, ArH).
(100 MHz, CDCl3) d: 19.15, 19.19, 33.95, 62.39, 71.41, 79.82, 103.65,
4.1.4. (Z)-4,4-Dimethyl-3-((trimethylsilyl)oxy)pent-2-enenitrile. A
solution of diisopropylamine (1.80 mL, 13.0 mmol) in THF (20 mL)
cooled to 0 ꢂC. n-BuLi (5.20 mL of a 2.5 M solution in hexanes,
13.0 mmol) was added dropwise and the reaction was stirred
a further 30 min at 0 ꢂC. The LDA solution was cooled to ꢀ78 ꢂC and
a solution of 4,4-dimethyl-3-oxopentanenitrile (1.25 g, 10.0 mmol)
in THF (5 mL) was added dropwise. After stirring at ꢀ78 ꢂC for an
additional 30 min, TMSCl (2.50 mL, 20.0 mmol) was added. The
reaction was allowed to slowly warm to rt and stirred an additional
3 h. The reaction was concentrated under reduced pressure and the
residue was taken up in hexanes, filtered through Celite eluting
with hexanes (100 mL), and solvents were removed under reduced
pressure. The orange oil was distilled under reduced pressure to
yield 1.14 g (58%, 102e103 ꢂC at 13 mbar) of the titled compound as
a clear oil (93:7 ratio of enol silane:ketone). This material is highly
moisture sensitive and must be stored at ꢄ4 ꢂC under an anhydrous
125.37, 140.05, 151.79, 153.47, 159.37, 176.85.
4.1.8. CAL catalyzed isolation of isobutyricacid-4-(R)-(2-amino-6-
choloropurin-9-yl)-[1,3]-dioxolan-2-(R)-yl methyl ester (11). The
a/
b
anomer mixture, 10 from the above coupling step was hydrolyzed
in 10 mL of pH 7.0 buffer (0.05 M potassium phosphate) and 10 mL of
THF and 300 mg of lipase acrylic resin from candida antarctica (CAL).
The mixture was stirred at room temperature for 20 h. After which
the a-isomer was hydrolyzed completely and 100 mL of CH2Cl2 was
added. The organic phase was collected and the aqueous layer was
extracted with CH2Cl2 (2ꢃ100 mL). The combined organic layer was
washed with water (50 mL), brine (50 mL) and dried over Na2SO4.
After removal of the solvent under reduced pressure, 11 was
obtained in 46% yield and used directly in the next step.
4.1.9. Preparation of (e)- -2-aminopurine dioxolane [(e)-APD]. A
D
atmosphere of nitrogen. 1H NMR (400 MHz, CDCl3)
1.09 (9H, s) 0.38 (9H, s). 13C NMR (100 MHz, CDCl3)
74.8, 38.3, 27.7, 1.2.
d
4.64 (1H, s)
solution of isobutyric acid-4-(R)(2-amino-6-chloro-purin-9-yl)-
[1,3]-dioxolan-2-(R)-yl methyl ester 11 (500 mg, 1.40 mmol), and
10% Pd/C (100 mg) in MeOH (50 mL) and NH4OH (5 mL) was kept
under hydrogen (1 atm) until the TLC showed disappearance of the
starting material (w24 h). The reaction mixture was degassed with
nitrogen, filtered through Celite and the solvent was removed un-
der reduced pressure. The residue was passed through a short flash
silica gel column (hexanes: ethyl acetate, 9:1 to 3:7 gradient) to
give the title compound (285 mg, 84%). Pure material was obtained
by crystallization in MeOH/CH2Cl2: mp: 155e156 ꢂC. LC-MS (EI):
d: 181.1, 118.5,
4.1.5. [(2R,4R)-[4-(2,6-Diamino-9H-purin-9-yl)-1,3-dioxolan-2-yl]
methanol
[(ꢀ)-DAPD]. (2R,4R)-2-Isobutyryloxymethyl-4-(2,6-
diaminopurin-9-yl)-1,3-dioxolane$2-propanol, 8 (31.1 g, 81.2 mmol)
was dissolved in 310 mL of NH3-saturated methanol. The solution
was stirred at 25 ꢂC for 15 h and the solvent was removed under
reduced pressure. The residue was crystallized from ethanol/water

L. Zhou et al. / Tetrahedron 68 (2012) 5738e5743
5743
238 (Mþ1). 1H NMR (400 MHz, DMSO-d6)
: 3.62 (dd, J¼6.0 Hz,
d
7. Narayanasamy, J.; Pullagurla, M. R.; Sharon, A.; Wang, J.; Schinazi, R. F.; Chu, C.
K. Antiviral Res. 2007, 75, 198.
8. Belleau, B. EP 0337713, 1989.
9. (a) Chu, C. K.; Schinazi, R. F. U.S. Patent 5179104, 1990. (b) Kim, H. O.; Schinazi,
R. F.; Nampalli, S.; Shanmuganathan, K.; Cannon, D. L.; Alves, A. J.; Jeong, S. J.;
Beach, J. W.; Chu, C. K. J. Med. Chem. 1993, 36, 30.
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T. L.; Rando, R. F. Nucleosides Nucleic Acids 1999, 18, 891.
J¼3.0 Hz, 2H, CH2); 4.23 (dd, J¼10.0 Hz, J¼5.5 Hz, 1H, CH); 4.54 (d,
J¼10.0 Hz, J¼1.5 Hz, 1H, CH); 5.07 (t, J¼3.0 Hz, 1H, OH); 5.15 (d,
J¼6.0 Hz,1H, CH); 6.33 (dd, J¼5.5 Hz, J¼1.5 Hz,1H, CH); 6.62 (bs, 2H,
NH2); 8.23 (s, 1H, CH); 8.60 (s, 1H, CH). 13C NMR (100 MHz, DMSO-
d6) d: 61.0, 70.4, 78.5, 105.5, 126.5, 140.0, 149.3, 152.6, 160.6. Cal-
11. Zottola, M. A.; Alonso, R.; Vite, G. D.; Fraser-Reid, B. J. Org. Chem. 1989, 54, 6123.
12. (a) Chu, C. K.; Ahn, S. K.; Kim, H. O.; Beach, J. W.; Alves, A. J.; Jeong, L. S.; Islam,
Q.; Van Roey, P.; Schinazi, R. F. Tetrahedron Lett. 1991, 32, 3791; (b) Kim, H. O.;
Ahn, S. K.; Alves, A. J.; Beach, J. W.; Jeong, L. S.; Choi, B. G.; Van Roey, P.; Schinazi,
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culated for C9H11N5O3$0.5H2O: C, 43.90; H, 4.91; N, 28.44. Found: C,
42.91; H, 4.80; N, 28.40.
Acknowledgements
13. Sznaidman, M. L.; Du, J.; Pesyan, A.; Cleary, D. J.; Hurley, K. P.; Waligora, F.;
Almond, M. R. Nucleosides, Nucleotides Nucleic Acids 2004, 23, 1875.
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WO2005074654, 2005.
Supported in part by NIH CFAR grant 2P30-AI-050409 and by
the Department of Veterans Affairs. Emory received no funding
from RFS Pharma, LLC to perform this work and vice versa.
COI: Dr. R.F.S. is the Founder of RFS Pharma, LLC, a major
shareholder of RFS Pharma, LLC and an inventor of DAPD/APD and
may receive royalties from the future sales of these drugs.
15. Popp, A.; Gilch, A.; Mersier, A.-L.; Petersen, H.; Rockinger-Mechlem, J.; Stohrer,
J. Adv. Synth. Catal. 2004, 346, 682.
€
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