Synthesis of the hepatitis B nucleoside analogue lagociclovir valactate

Article abstract of DOI:10.1021/op200153s

2′,3′-Dideoxy-3′-fluoro-5-O-[(S)-(+)-2-(l-valyloxy) -propionyl guanosine (lagociclovir valactate) is a prodrug of 3′-fluoro-2′,3′-dideoxyguanosine with high oral bioavailability in humans and potent activity against hepatitis B virus (HBV). A five-step synthesis of lagocyclovir valactate starting from 2-amino-6-chloropurine is described. The synthesis was performed at kilogram scale, and the target nucleoside prodrug was isolated as the hemisulphate salt with an overall yield of 23%. The major challenges were N-glycosylation of a 2-deoxyfluorosugar, which required separation of α- and β-anomers, and deprotection of the penultimate intermediate by hydrogenation.

Full text of DOI:10.1021/op200153s

ARTICLE
pubs.acs.org/OPRD
Synthesis of the Hepatitis B Nucleoside Analogue Lagociclovir Valactate
ꢀ
Martin Brodszki,† Birthe B€ackstr€om,† Karol Horvath,§ Torbj€orn Larsson,‡ Hakan Malmgren,†
Mikael Pelcman,‡ Horst W€ahling,‡ Hans Wallberg,‡ and Johan Wennerberg*,†
†DuPont Chemoswed, R&D, P.O. Box 839, SE-201 80 Malm€o, Sweden
‡Medivir AB, P.O. Box 1086, SE-141 22 Huddinge, Sweden
§KH Crystallization Service, Fornh€ojdsv€agen 56, SE-152 58 S€odert€alje, Sweden
ABSTRACT: 20,30-Dideoxy-30-fluoro-5-O-[(S)-(+)-2-(L-valyloxy)-propionyl] guanosine (lagociclovir valactate) is a prodrug of 30-
fluoro-20,30-dideoxyguanosine with high oral bioavailability in humans and potent activity against hepatitis B virus (HBV). A five-
step synthesis of lagocyclovir valactate starting from 2-amino-6-chloropurine is described. The synthesis was performed at kilogram
scale, and the target nucleoside prodrug was isolated as the hemisulphate salt with an overall yield of 23%. The major challenges were
N-glycosylation of a 2-deoxyfluorosugar, which required separation of R- and β-anomers, and deprotection of the penultimate
intermediate by hydrogenation.
’ INTRODUCTION
promoted by a catalytic amount of phosphoric acid. After
evaporation the crude product was partially hydrolyzed in
methanol/ammonia.6 Acetamide 3 precipitated from the solu-
tion upon concentration and was collected by centrifugation in
89% yield. The quality of the starting material was found to be a
critical factor in determining reaction outcome. 2-Amino-6-
chloropurine of 98% purity gave satisfactorily results, but batches
of 97% purity resulted in an approximately 15% lower yield. A
major drawback for this step was the large amounts of acetic
anhydride and ammonia/methanol required; unfortunately the
yield was highly dependent on a large excess of these reagents.
N-Glycosylation of 3 to yield 5 was achieved via silylation of 3
followed by reaction with protected 2-deoxyfluorosugar 67 and
trimethylsilyl trifluoromethanesulfonate (Scheme 1). Com-
pound 3 was silylated at two positions using 1,1,1,3,3,3-hexam-
ethyldisilazane and a catalytic amount of ammonium sulfate in a
mixture of 1,4-dioxane and diisopropyl ether (Figure 1) in order
to enhance the nucleophilicity of the N-9 nitrogen participating
in the glycosylation.8 The silylated intermediate was reacted with
2-deoxyfluorosugar 6 using a stoichiometric amount of trimethyl-
silyl trifluoromethanesulfonate (scheme 1). After workup the
product was obtained as a mixture of the R- and β-anomers in a
47/53 ratio. The difference in Rf value on silica gel TLC plates
was 0.1, and careful normal phase chromatography resulted in
the desired β-anomer 5 in 41% yield. All attempts to purify the
material by crystallization were unsuccessful. The glycosylation
mechanism should most likely be an SN1 reaction in which a
planar carbocation is formed from 2-deoxyfluorosugar 6 in the
rate-determining step. The silylated base can then attack from
both sides, which results in an equal or almost equal mixture of
anomers. This problem does not appear when using riboses
because neighboring group participation of the 2-acyl group
directs the incoming nucleophile from the top face, yield-
ing predominantly the β-anomer.9 It was noted that the pure
R-anomer of 2-deoxyfluorosugar 6 undergoes anomerization
Lagociclovir valactate (20,30-dideoxy-30-fluoro-5-O-[(S)-(+)-
2-(L-valyloxy)-propionyl] guanosine)1 (1), a prodrug of FLG (30-
fluoro-20,30-dideoxyguanosine) (2), has shown excellent pharma-
cokinetic properties in phase 1 clinical studies.2 After absorption,
lagociclovir valactate is converted to the nucleoside 2 which is
phosphorylated to the active metabolite FLG triphosphate. FLG
triphosphate is a competitive inhibitor which inhibits HBV DNA-
polymerase by competition with the natural substrate (deoxyguano-
sine triphosphate, dGTP) and causes chain termination. It has been
demonstrated in vitro that FLG exhibits similar inhibitory activity on
wild type as well as single- and multiple-drug-resistant HBV mutants
arising from adefovir and lamuvidine.3 Adefovir and lamivudine are
currently used in treatment of HBV, but both lead to the develop-
ment of a resistant virus, thereby losing their therapeutic usefulness.
The in vitro studies have indicated that the polymerase inhibitor
FLG blocks HBV polymerase by a different mechanism than
adefovir. FLG has also shown a profound antiviral effect after oral
administration in a Woodchuck model for Hepatitis B.4 Hepatitis B
is a common form of jaundice in need of better treatment alter-
natives than those currently available. FLG is also active against both
wild type and multiple-drug-resistant HIV-1, which has a polymer-
ase that resembles the HBV polymerase.5 To support clinical trials
kilogram quantities of 1 were required. The synthetic route devel-
oped consists of five synthetic steps followed by salt formation. The
most challenging steps were the N-glycosylation of 2-acetoamido-6-
chloropurine (3) and the deprotection of Cbz intermediate 4
(Scheme 1). In the N-glycosylation step both the R- and the desired
β-anomers were formed, which required careful separation. In the
deprotection step, the major challenge was to suppress catalyst
poisoning. The present method made it possible to manufacture 1at
kilogram scale in high purity whilst conforming to all regulatory
requirements.
’ RESULTS AND DISCUSSION
Commercially available 2-amino-6-chloropurine was first con-
Received: June 7, 2011
verted to its diacetamide, by treatment with acetic anhydride, and
Published: August 03, 2011
r
2011 American Chemical Society
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Scheme 1. Synthetic route to lagociclovir valactate
reaction conditions were examined (Table 1). It has been
reported that mild Lewis acids such as ZnCl2 and CuI have been
used in the synthesis of β-nucleoside anomers, but in these cases
chlorosugars were used as starting materials.9 Experiments with
ZnCl2 and CuI gave in our case no conversion of starting
material, and anomerization of 6 was not observed even after
18 h (entries 2 and 3); it was determined that ZnCl2 and CuI
were too weak as Lewis acids for the present application. BF3
etherate on the other hand gave 5 with an unfavorable ratio and
low conversion (entry 4). Chlorinated solvents have often been
applied in this type of reaction,10 but in our case the use of
dichloromethane favored the formation of the undesired R-
nucleoside anomer (entry 5). The same outcome was observed
for acetonitrile (entry 8). Solvents such as DMF, THF, benzene,
toluene, and methanol gave no conversion (entries 6, 7, 9ꢁ11).
Interestingly acetone gave a favorable R/β ratio but very low
conversion (entry 12). Eventually the large scale reaction was run
in 1,4-dioxane and diisopropyl ether with TMS-triflate at 20 °C
for 20 h.
We also attempted to apply R-halosugars as donors. This
reaction would follow an SN2 mechanism which would lead to
inversion of the stereochemistry. The conditions are mild; often
catalysts are unnecessary minimizing the risk for anomerization
of starting material. Many good results have been reported,11 but
our attempts failed. Although, it was possible to convert R-2-
deoxyfluorosugar 6 to the R-chlorosugar,12 it was not possible
to purify. The corresponding R-bromosugar was too labile to
be useful. Attempts were also made to use a mixture of R and
Figure 1. Bis-silylated 3 and starting materials 6 and 7.
during the reaction conditions. The observed anomerization was
very rapid. The conversion of the pure R-anomer to an anomeric
mixture required only a few minutes when the R-anomer
was treated with trimethylsilyl trifluoromethanesulfonate in 1,
4-dioxane. Anomerization also occurred with hydrogen chloride,
although at a slower rate. When R-2-deoxyfluorosugar 6 was
dissolved in 1,4-dioxane, without any acid added, anomerization
was hardly detected after 1 h. Regarding the kinetics, the R-2-
deoxyfluorosugar reacts more slowly than β-2-deoxyfluorosugar
and there is a competition between anomerization and glycosyla-
tion. It may also be that the use of pure R-2-deoxyfluorosugar is
not needed for this reaction (vide infra). Anyway, both the
anomerization of the starting material and the SN1 mechanism
in the glycosylation will affect the outcome of the reaction in a
negative way. The result of the reaction is dependent on the
choice of catalyst and solvent. Formation of the undesired R-
product 5 is favored by polar solvents, high temperature, and
strong Lewis acids; we also found that more diluted conditions
favored formation of the R-anomer. A number of different
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Table 1. Anomeric ratio for 5 under different reaction con-
Table 2. Reagents for coupling of 2 and 7a
ditions (selected experiments)a
Entry
Coupling reagent
Yield (%)
Purity (area%)
Temp Reaction
Conversionb
1
2
3
4
DCC
85
98ꢁ99b
95ꢁ98
very impure
98
Entry
Solvent
Dioxane
(°C) time (h) R/β Ratio
R + β (%)
Oxalyl chloridec
Pivaloyl chloride
EDC
52ꢁ95
32
1
21
21
21
21
21
21
21
21
21
21
21
21
8
22
18
18
22
7
47/53
ꢁ
89
2
Dioxane
no conversion
94ꢁ95
a All reactions were performed at small scale (2.5ꢁ12.5 mmol). b Chro-
matography was required. c Bad reproducibility and occasionally impure
due to side reactions.
3
Dioxane
ꢁ
no conversion
4
Dioxane
68/32
54/46
ꢁ
18
5
DCM
89
6
DMF
24
22
18
24
24
18
18
53
30
48
no conversion
7
THF
ꢁ
no conversion
required despite the apparent high purity (entry 1). Oxalyl
chloride showed poor reproducibility, and side reactions were
also observed (entry 2). Mixed anhydride formed with pivaloyl
chloride showed a consistently low yield (entry 3).
8
Acetonitrile
Benzene
58/42
ꢁ
73
9
no conversion
10
11
12
13
14
15
Toluene
ꢁ
ꢁ
no conversion
The Cbz-protecting group was removed from 4 by catalytic
hydrogenation with 10% palladium on carbon in a mixture of
ethyl acetate and acetic acid to give 8 (Scheme 1). This step
turned out to be very difficult; it was necessary to use large
amounts of the catalyst to get the reaction to go to completion.
However, a 40% reduction of the initial amount of catalyst
required was reached during laboratory development. The
amount of catalyst required was partly related to the purity of
4 obtained in the previous step. Although the starting material
had over 99% purity by HPLC, hydrogenation was sluggish. An
unknown impurity, not detected with HPLC, seemed to poison
the catalyst (vide supra), and treatment with activated charcoal
prior to hydrogenation provided no improvement. However,
purification of 4 by silica gel chromatography, in laboratory scale,
prior to hydrogenation gave a material which was easy to
hydrogenate. Reaction for 2 h at 2.7 bar at rt with 25% (w/w)
10% Pd/C gave full conversion. To examine if the impurity had
its origin in the 2-mercaptoethanol used to make 2, experiments
were performed in which 4 was treated with hydrogen peroxide
or Oxone in order to oxidize sulfur-containing impurities, prior
to a screening of hydrogenation conditions.
Palladium on charcoal, palladium black, and palladium hydro-
xide were tested with different solvents and solvent combina-
tions. Additionally catalysts with different palladium content
from different lots and suppliers and acidic conditions as well
as transfer hydrogenation conditions were investigated. To our
dismay the reactions were uniformly very slow or resulted in no
conversion of the starting material. As a comparison, 4 synthe-
sized from sulfur-free 2 also reacted very slowly under hydro-
genation conditions. Eventually, after crystallization from ethanol/
diethyl ether and repeated washings with diethyl ether, 4 suffi-
ciently pure for hydrogenation was obtained. This material
underwent hydrogenolysis with full conversion at 3 bar and
30 °C during 3 h. The major drawback for this procedure was that
nearly 50 mol % palladium was required to obtain full conversion,
but all further attempts to reduce the amount of palladium led to
incomplete conversion. On one occasion the reaction stalled at
60% conversion, and all attempts to continue the reaction by
addition of more catalyst failed. After careful examination of all
chemicals involved, it was discovered that, after evaporation of
“pure” ethyl acetate, an oily residue was obtained, which was the
likely culprit of catalyst poisoning. Laboratory experiments with
other batches of ethyl acetate confirmed this hypothesis. It is
worth mentioning that the impure ethyl acetate was approved
according to the actual specification. After the hydrogenolysis
was completed and the catalyst filtered off, the mixture was
Methanol
Acetone
no conversion
25/75
44/56
36/64
38/62
5
Dioxane + DCM
Dioxane + DCM
85
79
18
ꢁ4
Dioxane + DCM ꢁ13
a All reactions were performed with TMS-triflate except for entries 2, 3,
and 4 which were run with ZnCl2, CuI, and BF3 etherate, respectively.
b Conversion was measured with HPLC.
β-2-deoxyfluorosugar 6 as starting material. The yield was in the
same range as for the reaction with pure R-2-deoxyfluorosugar 6,
but the purity of the product was not satisfactory. Two impu-
rities, which were impossible to separate from 5 with chroma-
tography, were encountered. The present method made it
possible to produce kilogram quantities of 5 with good quality
in a yield of 41% (53% theoretically possible) with the low
yielding step early on in the synthetic sequence.
In the next step, 5 was treated with 2-mercaptoethanol and
sodium hydroxide resulting in cleavage of both protecting groups
and conversion of the 6-chloropurine to guanosine13 (Scheme 1).
After neutralization with acetic acid, compound 2 was isolated in
quantitative yield.
The Cbz-protected side chain 714 used in the next step was
supplied as the dicyclohexylamine salt, which was converted to
the free acid by treatment with hydrogen chloride in diethyl
ether. For the reaction of 2 and 7 (Scheme 1), N-(3-dimethyl-
aminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC) was
used as a coupling reagent in the presence of 1-hydroxybenzo-
triazole and dimethylaminopyridine in DMF. This combination
of reagents gave 4 with high reproducibility, in high yield and
purity. However, to obtain an optimal yield it was required to add
the coupling reagent in three portions and carefully monitor the
progress of the reaction with HPLC. After workup, the crude
material was crystallized from ethanol/diethyl ether, followed by
repeated washings with diethyl ether providing 4. This extensive
ether washing procedure was crucial to eliminate an unidentified
byproduct in the crude material that was otherwise found to
inhibit the subsequent hydrogenation. When the diethyl ether
wash residues were concentrated, the impurity was detected as a
discrete spot on TLC (Rf 0.67, n-heptaneꢁEtOAc 2:3). Despite
the tedious procedure the yield was fairly good (85%). Other
reagents were also tested including dicyclohexyl carbodiimide,
oxalyl chloride, and mixed anhydride. However, these reagents
were unsatisfactory in terms of purity, yield, and reproducibility
(Table 2). DCC gave a good yield, but chromatography was
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ARTICLE
treated with aqueous sodium sulfide in order to remove residual
palladium.15 A number of salt forms were screened in order to
find a salt with good water solubility. The hemisulfate of lago-
ciclovir valactate displayed a satisfactory water solubility of 9.8%
w/v at room temperature. The mixture containing 1 as the
acetate was treated with sulfuric acid in 2-propanol to give 1 as
the hemisulfate salt in 73% yield from 4. Analysis of the final
product revealed an assay typically around 97% with water and
2-propanol as the major contaminants. Other impurities found
were 2, ranging from 0.2 to 1.2%, and guanine sulfate, typically
0.5%. Despite using palladium catalysis in the penultimate step16
only 0ꢁ4 ppm palladium was detected when analyzed with AAS.
moist material (12.2 kg) was charged to a mixture consisting of
methanol (480 L) and ammonia (25%, 83 L). The mixture was
heated to 35 °C during 1 h and then cooled to 20 °C. The mixture
was transferred to another reactor via a cartridge filter after which
activated charcoal (Filtrasorb 400, 0.8 kg) was added. After
stirring for 1 h the mixture was filtered and the volume was
reduced via distillation at reduced pressure (15ꢁ20 mbar and not
over 45 °C) until less than 130 L remained.
The mixture was cooled to 20 °C. Centrifugation followed by
washing with water (15 L) and drying at reduced pressure
(8 mbar) at 50 °C gave the title compound (6.7 kg, 89%) as a
white solid: mp >300 °C; IR 3161 (broad), 1724, 1578, 1491,
1380 cmꢁ1; 1H NMR (DMSO-d6) δ 10.79 (s, 1H), 8.53 (s, 1H),
2.19 (s, 3H); 13C NMR (DMSO-d6) δ 168.4, 154.1, 151.9, 148.4,
145.0, 126.7, 24.4.
’ CONCLUSION
The highly water-soluble drug candidate lagociclovir valactate
(1) was synthesized in five steps in an overall yield of 23%.
The key N-glycosylation step resulted in a mixture of R- and
β-anomers which were separated by column chromatography.
Furthermore a problematic deprotection of the penultimate Cbz
carbamate by hydrogenolysis was carried out. The synthesis,
performed at kilogram scale, gave a material that fulfilled all
regulatory requirements.
9-(5-Pivaloyl-3-fluoro-2,3-dideoxy-β-D-ribofuranosyl)-2-
acetamido-6-chloropurine (5). In a glass-lined reactor charged
with 1,4-dioxane (28.3 L) were added under stirring 3 (1.9 kg, 9.0
mol), ammonium sulfate (20 g, 0.15 mol, finely grounded), and
1,1,1,3,3,3-hexamethyldisilazane (1.32 kg, 8.2 mol). The mixture
was heated gently to 96ꢁ99 °C and kept at reflux for 3 h
(CAUTION! foaming). 1,4-Dioxane (9 L) was distilled off after
which the mixture was cooled to 22ꢁ25 °C. To the mixture was
added diisopropyl ether (19 L) and 6 (1.0 kg, 4.3 mol) followed
by slow addition of trimethylsilyl trifluoromethanesulfonate
(1.0 kg, 4.5 mol). After stirring for 20 h at 20 °C, a mixture of
methanol (0.8 L) and triethylamine (0.8 L) was added at such a
rate that the temperature did not exceed 25 °C. The mixture was
cooled to 15 °C and then filtered through a glass-sintered funnel.
The filtrate was concentrated to a thick oil at reduced pressure
(10 mbar) and room temperature. Ethyl acetate (20 L) was
added, and the resulting solution was washed with aqueous acetic
acid (10%, 8 L) and aqueous potassium carbonate (10%, 8 L).
Silica gel 60 (0.035ꢁ0.070 mm) (3 kg) was added to the organic
phase, and the mixture was concentrated to dryness in an eva-
porator. The residue was purified with chromatography (silica gel
’ EXPERIMENTAL SECTION
General. HPLC analyses were performed using a YMC ODS-
A, 5 μ, 250 mm ꢂ 4.6 mm, 120 Å equipped with an SB-C18 guard
PAK. Elution conditions were employed with 20% acetonitrile in
aqueous ammonium acetate (3 mM). The flow rate was 1.0 mL/min,
and UV detection was at 220 nm for 1 and 254 nm for 2 and 5.
GC-analyses were performed using a J&W DB WAX, Bodman
part # 123-7033, 0.5 μm film thickness, 0.32 mm i.d., 30 m length.
Inlet temperature/detector temperature: 200/250 °C. Tempera-
ture gradient: 40 °C isothermal for 5 min and then ramped to
200 °C at profile 50 °C/min and held at 200 °C for 10 min.
Optical rotations were measured with a Perkin-Elmer 241. NMR
spectra were recorded at 300.14 MHz (proton) and 75.47 MHz
(carbon) respectively. Thin layer chromatography was per-
formed on Merck precoated TLC plates, which were visualized
with concentrated H2SO4 followed by heating. Solvents and
reagents were obtained from commercial sources and were used
as such without any further purification. All reactors used were
standard multipurpose equipment, either glass-lined or stainless
steel. All reactions in pilot-plant scale were for safety reasons
routinely carried out under an atmosphere of nitrogen.
2-Acetamido-6-chloropurine (3). In a stainless steel reactor
charged with acetic anhydride (168 kg, 1645 mol) was added,
under stirring, 2-amino-6-chloropurine (8.0 kg, 35.4 mol). The
mixture was heated to 135 °C, at which point a mixture of acetic
anhydride (5.5 kg, 48.6 mol) and phosphoric acid (85%, 34 g,
0.29 mol) was added. The mixture was refluxed for 75 min and
then cooled to 30 °C. A 140ꢁ150 L volume of acetic acid
anhydride was distilled off under reduced pressure (55ꢁ65 °C at
15ꢁ20 mbar). To the thick slurry was added methanol (480 L),
and the mixture was stirred for 14 h at 20ꢁ25 °C. Ammonia
(25%, 83 L) was added at such a rate that the temperature did not
exceed 43 °C. The mixture was cooled and stirred for 6 h at
22ꢁ26 °C. The volume in the reactor was reduced via distillation
at reduced pressure (15ꢁ20 mbar and not over 45 °C) until less
than 130 L remained. After cooling to 20 °C the mixture was
centrifuged, and the residue was washed with water (15 L). The
60, n-hexaneꢁethyl acetate, 2:3) which gave 0.728 kg (41%) of
21
the pure β-anomer as white crystals: Mp >300 °C; [R]D
=
+10.9 (c 2.3, CDCl3.); Rf 0.20 (n-heptaneꢁEtOAc 40:60); IR
3166 broad, 1725, 1580, 1429, 1383, 1229 cmꢁ1; H NMR
1
(CDCl3) 8.37 (dd, J1 = 8.2 Hz, J2 = 6.1 Hz, 1H), 8.15 (s, 1H),
5.36 (dd, J1 = 53.4 Hz, J2 = 5.0 Hz, 1H), 4.53 (dt, J1 = 24.0 Hz, J2 =
5.5 Hz, 1H), 4.49ꢁ4.32 (m, 2H), 3.16ꢁ2.79 (m, 2H), 2.48 (s,
3H), 1.27 (d, J = 2.0 Hz, 1H), 1.21 (s, 9H); 13C NMR (CDCl3) δ
178.1, 169.5, 151.8, 151.8, 151.6, 142.9, 128.8, 92.3 (d, J = 179
Hz), 85.4, 82.9 (d, J = 25 Hz), 62.9 (d, J = 10 Hz), 38.8, 37.5
(d, J = 21 Hz), 27.1, 25.1.
2,30-Dideoxy-30-fluoroguanosine (2). In a glass-lined reactor
charged with water (107 L) was added sodium hydroxide (1.1 kg,
27.5 mol), and the mixture was stirred for 15 min. 2-Mercap-
toethanol (1.2 kg, 15.4 mol) and 5 (2.184 kg, 5.28 mol) were
added, and the mixture was heated to reflux. After 1.5 h at reflux,
the mixture was cooled to 40ꢁ45 °C and acetic acid (1.7 kg, 28.3
mol) was added slowly over 1 h. The mixture was cooled to
0ꢁ4 °C and stirred at this temperature for 2 h. The crude
product was isolated by filtration. Washing of the filtrate with
cold (0ꢁ4 °C) water (20 L) and cold (0ꢁ4 °C) ethanol (5 L)
followed by drying at reduced pressure (20 mbar) at 45ꢁ55 °C
gave the title compound as white crystals (1.42 kg 100%):
21
Mp 242 °C (dec.); [R]D = ꢁ38.1 (c 0.53, DMF); IR 3166
1
(broad), 1726, 1636, 1596, 1543, 1487, 1401, 1056 cmꢁ1; H
NMR (DMSO-d6) δ 10.69 (s, 1H), 7.95 (s, 1H), 6.49 (s, 2H),
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6.14 (dd, J1 = 5.6 Hz, J2 = 9.2 Hz, 1H), 5.38 (dd, J1 = 3.9 Hz, J2 =
46.3 Hz, 1H), 5.16 (t, J = 5.4, 1H), 4.16 (dt, J1 = 4.7 Hz, J2 =
26.9 Hz, 1H), 3.56 (m, 2H), 2.92ꢁ2.53 (m, 2H); 13C NMR
(DMSO-d6) δ 161.9, 158.9, 156.2, 140.5, 121.9, 99.0 (d, J =
173 Hz), 90.2 (J = 22 Hz), 87.8, 66.1 (J = 11 Hz), 41.9 (J = 20 Hz).
(S)-(+)-2-(N-Cbz-L-valyloxy)propionic acid (7). In a glass-
lined reactor charged with ethyl acetate (90 L) was added 7 as the
dicyclohexylammonium salt12 (6.0 kg, 11.9 mol) with stirring.
Hydrochloric acid in diethyl ether (1.0 M, 12.9 L, 12.9 mol) was
added during 30 min. The mixture was passed through a filter,
which was washed with ethyl acetate (42 kg). The solution was
concentrated by distillation under reduced pressure (20 mbar).
Approximately 130 L were distilled off, and the remaining thick
oil was used directly in the next step.
20,30-Dideoxy-30-fluoro-5-O-[(S)-(+)-2-(L-valyloxy)-propio-
nyl]guanosine Hemisulfate (1), Lagociclovir Valactate. In a
hydrogenation reactor charged with ethyl acetate (130 L kg) and
acetic acid (45 L) was added 4 (4.53 kg, 7.9 mol). The mixture
was warmed to 26ꢁ30 °C after which 10% palladium on charcoal
(3.9 kg, 3.6 mol, 46 mol %) was added. The mixture was hydro-
genated at 3 bar and 30 °C for 3 h. The progress of the reaction
was checked with TLC and HPLC. The catalyst was filtered off,
and the filter was rinsed with a 1:3 mixture of ethyl acetate and
acetic acid (40 L). The combined organic phases were concen-
trated via distillation at reduced pressure (55ꢁ60 °C jacket
temperature, 20 mbar) until 4ꢁ5 L remained. Water (4.4 L) was
added to the oil and stirred at 22ꢁ25 °C for 15 min. Aqueous
sodium sulfide (50%, 0.5 kg) was added, and the mixture was
then stirred for 5 min followed by 15 min without stirring. A
suspension of charcoal (40 g) in water (100 mL) was added and
stirred for 20 min. The mixture was filtered, and the filtrate was
transferred to a glass-lined reactor to which 2-propanol (5.4 kg)
was then added. Aqueous sulfuric acid (20%, 2.47 kg, 5.04 mol)
was added until the pH was 2.2ꢁ2.3. 2-Propanol (14 L) was
added over 15 min with stirring. After 30 min the pH was
adjusted to 2.2ꢁ2.3 by the addition of aqueous sulfuric acid
(150 g) after which the mixture was stirred for another 2.5 h. The
product was isolated via filtration and then washed with a 3:1
mixture of 2-propanol and water (3.4 L) and finally with
2-propanol (9 L). Drying at 50 °C under reduced pressure (8 mbar)
for 8 h gave the title compound as a white powder. Yield 3.1 kg
(73%) as hemisulfate.
20,30-Dideoxy-30-fluoro-50-O-[(S)-(+)-2-(N-Cbz-L-valyloxy)-
propionyl]guanosine (4). Compound 7 was dissolved in DMF
(170 L) and transferred to a glass-lined reactor. To the reactor
were added 2 (2.5 kg, 9.3 mol), 1-hydroxybenzotriazole (1.5 kg,
11.1 mol), 4-(dimethyl)aminopyridine (0.23 kg, 1.9 mol), and
DMF (293 L) with stirring. DMF (230 L) was distilled off at
reduced pressure (50ꢁ60°, 20 mbar) after which the mixture was
cooled to 25ꢁ28 °C. N-(3-Dimethylaminopropyl)-N0-ethylcar-
bodiimide hydrochloride (EDC) (1.7 kg, 8.9 mol) was added,
and the mixture was stirred for 5 h at 22ꢁ25 °C. Another portion
of EDC (1.1 kg, 5.7 mol) was added, and stirring was continued
for 14 h at 22ꢁ25 °C. Finally a small portion EDC (0.2 kg, 1.0 mol)
was added, the mixture was heated to 42ꢁ46 °C and kept at this
temperature for 1 h. The progress of the reaction was monitored
with HPLC. DMF (227 L) was distilled off under reduced
pressure (50ꢁ60 °C, 20 mbar) until a thick oil remained. Ethyl
acetate (140 L) and water (60 L) were added, and the mixture
was stirred for 15 min at ambient temperature. After phase sepa-
ration the aqueous phase was extracted with ethyl acetate (90 L).
The combined organic phases were washed with saturated
aqueous sodium bicarbonate (2 ꢂ 74 L), water (15 L), 5%
aqueous acetic acid (2ꢂ74 L), and again with water (15 L). Ethyl
acetate was distilled off under reduced pressure (33ꢁ35 °C
jacket temperature, 20 mbar) until a thick oil remained. Toluene
(15 kg) was added to the oil and then distilled off under vacuum
(40ꢁ45 °C jacket temperature, 20 mbar). To the remaining
slurry was added ethanol (70 kg), and the mixture was heated to
75ꢁ78 °C and kept at this temperature for 10 min. Ethanol
(50 L) was distilled off under reduced pressure (30ꢁ35 °C jacket
temperature, 20 mbar), after which the mixture was cooled to
30ꢁ33 °C and diethyl ether (60 kg) was added. The mixture was
cooled during 2 h to 0ꢁ4 °C and kept at this temperature for
another 2 h. The resulting precipitate was isolated on a filter and
washed with diethyl ether (10 kg). The moist material was mixed
with diethyl ether (110 kg), stirred for 2 h, and isolated via
filtration. This procedure was repeated one more time with 110 L
diethyl ether and one time with 78 L of diethyl ether. After final
filtration the residue was dried at 50 °C under reduced pressure
(8 mbar) to give 4.56 kg (85%) of the title compound as a beige
powder. [R]D23 = ꢁ9.8 (c 0.60, EtOAc); mp 126ꢁ127 °C; IR 3150
(broad), 1752, 1702, 1633, 1595, 1517, 1090; 1H NMR (DMSO-
d6) δ 7.92 (s, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.36 (m, 5H), 6.52
(s, 2H), 6.18 (dd, J1 = 8.9 Hz, J2 = 5.8 Hz,1H), 5.58ꢁ5.44 (m, 1H),
5.12 (m, 1H), 5.04 (s, 2H), 4.44ꢁ4.20, (m, 3H), 4.03 (m, 1H),
3.06ꢁ2.55 (m, 2H), 2.10 (m, 1H), 1.40 (d, J = 7.0 Hz, 3H), 0.92
(t, J = 7.0 Hz, 6H); 13C NMR (DMSO-d6) δ 171.2, 169.9, 156.6,
156.5, 153.7, 151.0, 135.3, 128.3, 127.8, 127.7, 116.9, 95.5, 82.7, 81.7,
81.5, 68.6, 65.5, 59.6, 29.7, 18.8, 17.6, 16.7.
21
[R]D = +0.77 (c 1.6, H2O); IR 3164 (broad), 1742, 1687,
1636, 1598, 1532, 1485, 1402, 1056 cmꢁ1; 1H NMR (DMSO-d6)
δ 7.92 (s, 1H), 6.62 (s, broad, 2H), 6.20 (q, J = 6.0 Hz, 1H), 5.42,
(dd, J1 = 53.5 Hz, J2 = 3.8 Hz, 1H), 5.15 (q, J = 6.9 Hz, 1H),
4.42ꢁ4.25 (m, 3H), 3.08ꢁ2.85 (m, 1H), 2.19ꢁ2.08 (m, 1H),
1.44 (d, J = 7.0 Hz, 3H), 1.04 (d, J = 6.1 Hz, 1H), 0.96 (s, 6H);
13C NMR (DMSO-d6) δ 169.7, 168.4, 156.8, 153.8, 151.1, 135.5,
116.9, 92.8 (d, J = 175 Hz), 82.7 (d, J = 10 Hz), 81.5 (d, J = 25
Hz), 69.5, 64.3, 57.8, 36.0 (d, J = 20 Hz), 29.1, 25.5, 17.6, 16.7.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: johan.wennerberg@swe.dupont.com.
’ ACKNOWLEDGMENT
We are grateful to Johan Eriksson-Bajtner for help with NMR
experiments; to Anna Castensson for assistance with analyses; to
Carl-Magnus Andersson for valuable comments on the manuscript,
and to Hans Uvelius and Roger Johnsson for running the pilot plant.
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