Development of a practical synthesis of a purine nucleoside phosphorylase inhibitor: BCX-4208

Article abstract of DOI:10.1021/op9001142

A practical synthesis of the purine nucleoside phosphorylase (PNP) inhibitor BCX-4208 (1) was accomplished in three telescoped steps. Mannich condensation of the 4-benzyloxy-9-deazahypoxanthine with (3R,4R)-3-hydroxy-4- (hydroxymethyl)pyrrolidine and formaldehyde followed by removal of the protecting group and crystallization furnished the desired product as a hydrochloride salt in 85% overall yield and 99.8% purity. A scalable synthesis of 9-deazahypoxanthine is also reported. 2009 American Chemical Society.

Full text of DOI:10.1021/op9001142

Organic Process Research & Development 2009, 13, 928–932
Development of a Practical Synthesis of a Purine Nucleoside Phosphorylase
Inhibitor: BCX-4208
Vivekanand P. Kamath,* Jesus J. Juarez-Brambila, Christopher B. Morris, Christopher D. Winslow, and Philip E. Morris, Jr.
BioCryst Pharmaceuticals, Inc., 2190 Parkway Lake DriVe, Birmingham, Alabama 35244, U.S.A.
Abstract:
A practical synthesis of the purine nucleoside phosphorylase (PNP)
inhibitor BCX-4208 (1) was accomplished in three telescoped steps.
Mannich condensation of the 4-benzyloxy-9-deazahypoxanthine
with (3R,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine and form-
aldehyde followed by removal of the protecting group and
crystallization furnished the desired product as a hydrochloride
salt in 85% overall yield and 99.8% purity. A scalable synthesis
of 9-deazahypoxanthine is also reported.
Results and Discussion
The retrosynthetic analysis of 1 suggested that the molecule
could be assembled by the formal condensation of formalde-
hyde, (3R,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine HCl (3)
Introduction
and 9-deazahypoxanthine (2) (see Scheme 1). This approach
The enzyme purine nucleoside phosphorylase (PNP) cata-
lyzes the cleavage of deoxyribonucleosides to the corresponding
purine base and sugar-1-phosphate. Children who are deficient
in PNP suffer from T-cell immunodeficiency due to the
accumulation of dGTP which prevents the proliferation of
T-lymphocytes.1-3 Hence, inhibitors against PNP are immu-
nosuppressive in nature and are active against T-cell malignan-
cies and T-cell disorders.4 The discovery of potent PNP
inhibitors was based on studies by Schramm and co-workers
on transition state analysis of the human PNP.5 The first
generation of PNP inhibitors includes forodesine HCl, a
compound currently in clinical trials at BioCryst Pharmaceu-
ticals Inc. for relapsed/resistant T-cell leukemia.6 A second-
generation transition state analogue has been identified as BCX-
4208 (1) whose primary advantage is that it binds more tightly
to human PNP.7a,b This molecule was prepared by Schramm,
Tyler, and co-workers from whom BioCryst acquired the rights.
While this was an excellent synthesis for small-scale work,
multikilogram quantities required a new process. Herein is
described our development of a practical synthesis of 1 and
9-deazahypoxanthine (2).
was successful.7c
Synthesis of 9-Deazahypoxanthine (2). There have been
relatively few syntheses of the pyrrolo[3,2-d]pyrimidine ring
system. Two early procedures suffered from either low yields
or purification challenges and were unlikely to provide scalable
processes.8-10 A recent report11 describing a direct synthetic
route by the condensation of isoxazole and diethyl aminoma-
lonate, appeared initially promising. However, attempts to repeat
the process on a large scale led to problems for sourcing
isoxazole, monitoring the reaction, workup, stability of inter-
mediates, and reproducibility. This prompted the search for an
alternate route.
A report published12 in 1996 led directly to our multikilo-
gram process as shown in Scheme 2. The pyrrole 5 could be
synthesized by refluxing ethyl (ethoxymethylene)cyanoacetate,
diethyl aminomalonate, and sodium methoxide in methanol for
24 h. Neutralization of the reaction to ∼pH 7 was carried out
by addition of acetic acid. This was followed by evaporation
of the solvent (∼65% of the original volume) to furnish a slurry.
Addition of water followed by rapid agitation of the slurry
produced 5 in 79% yield and in sufficient purity to be carried
directly to the next step. Condensation of 5 with formamidine
acetate in ethanol under reflux conditions for 20 h, followed
by addition of water to the hot solution resulted in the
precipitation of 6 in 72% yield.
* Author to whom correspondence may be sent. E-mail: vkamath@biocryst.com.
Telephone: (205)-444-4600. Fax: (205)-444-4640.
(1) Horenstein, B. A.; Parkin, D. W.; Estupinan, B.; Schramm, V. L.
Biochemistry 1991, 30, 10788.
(2) Horenstein, B. A.; Schramm, V. L. Biochemistry 1993, 32, 7089.
(3) Miles, R. W.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.; Parkin,
D. W.; Schramm, V. L. Biochemistry 1999, 38, 13147.
(4) Markert, M. L. Immunodefic. ReV. 1991, 3, 45.
The final step was the base-catalyzed decarboxylation of
compound 6. A series of experiments with different bases were
attempted, and 10% KOH solution gave the best results.13-16
(5) (a) Lewandowicz, A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.;
Schramm, V. L. J. Biol. Chem. 2003, 278, 314651. (b) Miles, R. W.;
Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L.
Biochemistry 1998, 37, 8615.
(8) Imai, K. Chem. Pharm. Bull. 1964, 12, 1030.
(9) Klein, R. S.; Lim, M. I.; Tam, S. Y. K.; Fox, J. J. J. Org. Chem.
1978, 43, 2536.
(6) (a) Furneaux, R. H.; Limberg, G.; Tyler, P. C.; Schramm, V. L.
Tetrahedron 1997, 53, 2915. (b) Furneaux, R. H.; Schramm, V. L.;
Tyler, P. C. Bioorg. Med. Chem. 1999, 7, 2599.
(10) (a) Taylor, E. C.; Young, W. B.; Ward, C. C. Tetrahedron Lett. 1993,
34, 4595. (b) Taylor, E. C.; Young, W. B. J. Org. Chem. 1995, 60,
7947.
(7) (a) Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V. L.;
Tyler, P. C. J. Med. Chem. 2003, 46, 5271. (b) Lewandowicz, A.;
Shi, W.; Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Basso, L. A.;
Santos, D. S.; Almo, S. C.; Schramm, V. L. Biochemistry 2003, 42,
6057. (c) Evans, G. B.; Furneaux, R. H.; Tyler, P. C.; Schramm, V. L.
Org. Lett. 2003, 5, 3639.
(11) Furneaux, R. H.; Tyler, P. C. J. Org. Chem. 1999, 64, 8411.
(12) Elliott, A. J.; Montgomery, J. A.; Walsh, D. A. Tetrahedron Lett. 1996,
37, 4339.
(13) Brown, B. R. Q. ReV. 1951, 5, 131.
928
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10.1021/op9001142 CCC: $40.75  2009 American Chemical Society
Published on Web 07/24/2009

Scheme 1. Retrosynthetic analysis of BCX-4208 (1)
Scheme 3. Synthesis of
(3R,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine HCl (3)
Scheme 2. Synthesis of 9-deazahypoxanthine (2)
Scheme 4. Synthesis of BCX-4208 (1) via Mannich
condensation
Our optimized process consisted of refluxing 6 in 10% KOH
solution for 40 h, followed by neutralization with acid to isolate
2 in 90% yield. The synthesis of 9-deazahypoxanthine (2) was
thus accomplished in three steps in 51% overall yield and is
currently in use for the synthesis of multikilogram quantities.
Synthesis of (3R,4R)-3-Hydroxy-4-(hydroxymethyl)pyr-
rolidine HCl (3). The synthesis of the western section of 1,
3-hydroxy-4-(hydroxymethyl)pyrrolidine HCl (3) was first
prepared by Biel and co-workers17 from N-benzyl glycinate and
ethyl acrylate as a mixture of cis/trans isomers. The synthesis
of pure enantiomer (3R,4R)-3-hydroxy-4-(hydroxymethyl)pyr-
rolidine HCl (3) was first made from D-xylose over several steps
but in low yields by Pedersen and co-workers.18
Asymmetric 1,3-dipolar cycloaddition of an azomethine ylide
and camphor sultam auxiliary gave 3, but was unsuitable for
large-scale operation as the camphor sultam auxiliary was very
expensive.19 An elegant synthesis by Tyler and co-workers for
the synthesis of 3 has been published.20 Nitrone cycloaddition
to diethyl maleate, followed by resolution of the desired isomers
by enzymatic transformation, gave the desired product in
excellent yields. As shown in Scheme 3, the 1,3-cycloaddition
reaction of nitrone 8 (formed in situ) to diethyl maleate
proceeded to give the diester cyclized compound 9. Reductive
cleavage of the N-O bond with zinc and acetic acid furnished
a racemic mixture of ꢀ-amino alcohol, 10. A number of
enzymes were screened for effective resolution. Among them,
Novozyme 435 gave the best results in terms of yield and
enantiomeric purity. The compounds 11 and 12 were separated
efficiently by extraction using ethyl acetate. Reduction of the
carboxylic acid using borane generated in situ from sodium
borohydride and BF3 ·Et2O in THF gave 13 as a crystalline
compound. Hydrogenolysis of 13 furnished 3 as a light-orange
syrup. The new process for the synthesis of 3 had fewer
transformations than previously known methods. The method
was scaled up to multikilogram quantities by BioCryst without
any further modifications to the process.
Initial Approach to BCX-4208 (1). The Mannich reaction
provides a direct route for the formation of carbon-carbon and
carbon-nitrogen bonds.21 Tyler and co-workers have reported
the synthesis of several analogues of 1 using the Mannich
reaction.7c A mixture of (3R,4R)-3-hydroxy-4-(hydroxymeth-
yl)pyrrolidine HCl (3), sodium acetate, water, aqueous form-
aldehyde, and 9-deazahypoxanthine (2) was heated. The re-
sulting solid residue produced after workup was purified by
column chromatography and converted to the hydrochloride salt
in 35% yield. Although 1 could be synthesized by this one-
step operation, the process was not amenable to scale up as it
involved column chromatography and the active pharmaceutical
ingredient (API) was contaminated with process impurities
resulting in only a 35% yield (see Scheme 4).
The low yields for the Mannich reactions could possibly due
to the compatibility of the two starting materials. Compound 2
can be dissolved with great difficulty in hot water. Hence, the
Mannich reaction had to be carried out under reflux conditions
in water, thereby resulting in the formation of process impurities.
These have been identified as 9-hydroxyl 9-deazahypoxanthine,
C-9 methylene-bridged dimer of 2, and unreacted 2.
Process Development of BCX-4208, (1). A number of
derivatives of 2 were synthesized by introduction of protecting
groups at the C-4 position of 2. Among those included the
widely used methyl and the benzyl groups. The methyl group
(14) Sultanov, A. S. J. Gen. Chem. (USSR). 1946, 16, 1835.
(15) Chen, T. C.; Yan, S. J.; Wang, C. H. Chem. Ind. 1970, 895.
(16) Winslow, C. D.; Morris, P. E., Jr. Carboxy pyrrole, process of
preparing and use as precursor. U.S. Patent 6,972,331, 2005; CAS
# 5655-01-6.
(17) Jaeger, E.; Biel, J. H. J. Org. Chem. 1965, 30, 740.
(18) Filichev, V. V.; Brandt, M.; Pedersen, E. B. Carbohydr. Res. 2001,
333, 115.
(19) Karlsson, S.; Hogberg, H.-E. Tetrahedron: Asymmetry 2001, 12, 1977.
(20) Clinch, K.; Evans, G. B.; Furneaux, R. H.; Lenz, D. H.; Mason, J. M.;
Mee, S. P. H.; Tyler, P. C.; Wilcox, S. J. Org. Biomol. Chem. 2007,
5, 2800.
(21) Modnikova, G. A.; Sizova, O. S.; Novitskii, K. Y.; Ryabokon, N. A.;
Vlasova, T. M.; Chernov, V. A. Khim.-Farm. Zh. 1982, 16, 548.
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Scheme 5. Improved synthetic route to make 4-benzyloxy
mentation with different catalysts showed that the hydrogenation
step was sensitive to catalyst-poisoning agents. This problem
was overcome by a charcoal treatment of the solution prior to
hydrogenation. The reaction was observed to be complete in a
short period of time using absolute ethanol and at higher
temperature. Compound 13 was dissolved in absolute ethanol
and passed through an activated charcoal filter. Hydrogenation
was carried out using 10% Pd/C catalyst. The reaction was
complete in 5 h as compared to 24 h on kilo scale. The catalyst
was filtered off and the filtrate telescoped to the next step. The
Mannich reaction was explored by varying the time and
temperature. It was concluded that longer reaction times led to
slight improvement in the yield without affecting the formation
of any new side products. The reaction proceeded to completion
in 6 h (as compared to 3 h on kilo scale). The final hydrogena-
tion step was optimized by adjusting the concentration with
ethanol/water, and the reaction was carried out using 10% Pd/C
catalyst. The catalyst was filtered at 40-50 °C to facilitate rapid
filtration. The filtrate was distilled under vacuum to ∼40% of
the original volume to furnish a suspension of the crude product
that was filtered and dried. The crude product was suspended
in a mixture of ethanol/water and heated to reflux with the
addition of 32% hydrochloric acid. The suspension was cooled
to ambient temperature and stirred for an additional 3 h to
precipitate out the product. Crystallization of the crude API in
water/ethanol mixture furnished crystals of BCX-4208 (1) in
yields comparable to the kilo-scale process.
9-deazahypoxanthine (15) and BCX- 4208 (1)
was introduced by reacting 14 with 1 M sodium methoxide in
methanol under reflux conditions. Mannich coupling under
standard conditions, followed by deprotection of the methoxy
group with 37% hydrochloric acid and methanol (1:1, v/v) under
reflux conditions resulted in formation of a unstable dihydro-
chloride salt. Introduction of a benzyl group at the C-4 position
would eliminate this problem, thereby allowing a handle on
the outcome of the final product. Accordingly, 2 was treated
with neat phosphorous oxychloride to produce 14 as a yellow
solid in 97% yield after workup.22 This was in turn treated with
1 M sodium benzyloxide solution at reflux to furnish 15 as a
tan solid in 65% yield. While this required the addition of a
protection/deprotection sequence to the process, the overall
improvements in yield and processability were deemed a good
trade-off.
Conclusion
An improved method for the manufacture of BCX-4208,
(1) has been developed. The process was streamlined with the
help of three telescoped steps without any column chromatog-
raphy or isolation of the intermediates to furnish the desired
product in excellent yields. The process has been successfully
scaled up to 25 kg batches for use in toxicology studies and
ongoing phase I/II clinical trials.
Experimental Section
Once 13 was hydrogenated using Pd(OH)2/C in ethanol to
furnish 3, the catalyst was filtered over a Celite pad, and the
filtrate was telescoped to the next step. The reaction mixture
was heated followed by addition of aqueous formaldehyde and
15. Upon completion of the reaction, the mixture was cooled,
and the filtrate (containing product 16) was telescoped to the
next step. The filtrate was presaturated with ammonia and
hydrogenated over Pd(OH)2/C to give 17 as a solid following
workup. Following the production of the HCl salt 1 in aqueous
ethanol by the addition of 37% hydrochloric acid and
recrystallization from the same solvent mixture, crystals
of the API were isolated in 85% yield and 99.8 wt %
purity (see Scheme 5).
General. The samples were analyzed using HPLC run on a
Hewlett-Packard system, model no. HP-1100 with a photodiode
array detector. Analytical HPLC conditions were: Zorbax SB-
C3 150 mm × 4.6 mm and Zorbax SB-C3 250 mm × 4.6 mm
plumbed in series. Mobile phase A: 0.05 M formic acid, B:
acetonitrile. Gradient from 0 to 13% B for 12 min; 13-50% B
for 6 min; hold 50% B for 4 min; 0% B for 2 min; hold 100%
A for 2 min; post time 10 min; flow rate: 1.3 mL/min; 25 °C.
3-Amino-1H-pyrrole-2,4-dicarboxylic Acid Dimethylester
(5). Sodium methoxide (9.18 L, 42.5 mol) was added to a
solution of methanol (20 L) and diethyl aminomalonate
hydrochloride (3.0 kg, 14.1 mol) followed by ethyl (ethoxym-
ethylene)cyanoacetate 4 (2.40 kg, 14.1 mol) added over 1 h at
e45 °C. The mixture was refluxed for 24 h and then cooled to
ambient temperature. The reaction mixture was neutralized by
addition of glacial acetic acid (1.62 L, 28.3 mol), and solvent
(∼55%) was distilled to furnish a slurry. Water (15 L) was
added to the slurry followed by strong agitation. The slurry was
filtered, and the solids were washed with water (5 L) to furnish
Optimized Process Route-Pilot Scale. The kilo-scale
process was examined for further improvements prior to scale-
up in the pilot plant. The first step of the process required a
long reaction time for complete hydrogenation and used
inconveniently large volumes of ethanol and water. Experi-
(22) Evans, G. B.; Furneaux, R. H.; Hutchison, T. L.; Kezar, H. S.; Morris,
1
P. E.; Schramm, V. L.; Tyler, P. C. J. Org. Chem. 2001, 66, 5723.
5 (2.23 kg, 79%) as a tan solid. Mp 165 -166 °C; H NMR
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(300 MHz, DMSO-d6): δ 11.66 (bs, 1 H), 7.26 (s, 1 H), 5.64
(s, 2 H), 3.74-3.72 (2 × s, 6 H); 13C NMR (75 MHz, DMSO-
d6): δ 165.3, 161.3, 126.7, 103.9, 101.8, 73.7, 50.8, 50.7; IR
νmax 3491, 3265, 1676 cm-1. Elem. anal.: calculated for
C8H10N2O4: C, 48.46; H, 5.08; N, 14.13, found: C, 48.49; H,
5.13; N, 14.15.
4-Oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidine-7-car-
boxylic Acid Methyl Ester (6). Formamidine acetate (4.70 kg,
45.1 mol) was added to a solution of compound 5 (2.20 kg,
11.2 mol) in ethanol (20 L), and the reaction was refluxed for
20 h. Water (5 L) was added to the hot solution, and the
resulting slurry was filtered to furnish 6 (1.57 kg, 72%) as a
tan solid. Mp 220-222 °C; 1H NMR (300 MHz, DMSO-d6):
δ 7.97-7.96 (2 × s, 2 H), 3.73 (s, 3 H); 13C NMR (75 MHz,
DMSO-d6): δ 163.5, 154.4, 144.8, 143.7, 132.7, 119.6, 112.1,
51.1; IR νmax 3432, 2902, 1676 cm-1. Elem. anal.: calculated
for C8H7N3O3: C, 49.72; H, 3.65; N, 21.76, found: C, 50.02;
H, 3.59; N, 21.72.
3,5-Dihydropyrrolo[3,2-d]pyrimidin-4-one (2). Compound
6 (1.08 kg, 5.67 mol) was refluxed with 10% aqueous KOH
(11 L, 22.5 mol) for 40 h. The reaction mixture was cooled to
25-35 °C and then neutralized to pH 6.5-7.5 with addition
of glacial acetic acid (1.32 L, 22.5 mol) with gas evolution.
The precipitate was filtered and washed with water (5 L). The
solid was dried in Vacuo at 80 °C to furnish 2 (688.5 g, 90%)
as a solid. Mp 260-262 °C; 1H NMR (300 MHz, DMSO-d6):
δ 12.05 (bs, 1 H), 11.82 (bs, 1 H), 7.77 (s, 1 H), 7.36 (s, 1 H),
6.35 (s, 1 H); 13C NMR (75 MHz, DMSO-d6): δ 153.8, 144.8,
141.6, 127.5, 117.9, 103.1; IR νmax 3109, 3041, 1674 cm-1.
Elem. anal.: calculated for C6H5N3O: C, 53.33; H, 3.73; N,
31.10, found: C, 53.35; H, 3.73; N, 31.06.
15 (515 g, 65%) as a tan-colored powder. Mp 158-160 °C;
1H NMR (300 MHz, DMSO-d6): δ 8.33 (s, 1 H), 7.20-7.70
(m, 6 H), 6.57 (d, J ) 3.6 Hz, 1 H), 5.58 (s, 2 H); 13C NMR
(75 MHz, DMSO-d6): δ 154.8, 150.3, 148.3, 136.6, 130.4,
128.4, 128.1, 128.0, 114.1, 101.6, 66.8; IR νmax 3128, 2979,
1621 cm-1. Elem. anal.: calculated for C13H11N3O: C, 69.35;
H, 4.92; N, 18.66, found: C, 69.30; H, 4.92; N, 18.36.
(3R,4R)-3-Hydroxy-4-(hydroxymethyl)pyrrolidine (3). A
mixture of 13 (45 kg, 217 mol), activated charcoal (2.25 kg),
and ethanol (110 L) was stirred at 20-25 °C for 0.5 h and
pressure filtered. The filtrate, 10% Pd/C catalyst (7.6 kg), and
ethanol (15.7 kg) were pressurized to 5 bar with hydrogen gas
and heated to 55-60 °C. The reaction mixture was stirred for
5 h until there was no more uptake of hydrogen gas. Upon
completion of the reaction (<5% of 13 remaining by HPLC)
the reactor was cooled to 20-25 °C. The pressure was released
from the vessel, which was then flushed several times with
nitrogen. The contents of the vessel were filtered under pressure
through a lenticular filter, and the filtrate (303 kg, containing
3) was telescoped to the next step.
(4R)-1-((4-Benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-
methyl)-4-(hydroxymethyl)pyrrolidin-3-ol (16). The reactor
was charged with the ethanolic solution of 3 (303 kg of ethanol
containing ∼25.4 kg of 3). Ethanol (∼220 L) was distilled off
at reduced pressure (1.5 bar). Water (80 kg) was charged
followed by the addition of 15 (42.5 kg, 189 mol) and then
37% aqueous formaldehyde solution (21.6 kg, 266 mol). The
contents were heated to 55-60 °C for 6 h. Upon completion
of the reaction (<5% of 15 remaining by HPLC) the reaction
mixture was cooled to 20-25 °C followed by the addition of
activated charcoal (1.5 kg), and the mixture stirred for 30 min.
The contents were filtered under pressure through a lenticular
filter, and the filtrate (412 kg) was telescoped to the next step.
7-(((4R)-3-Hydroxymethyl)pyrrolidin-1-yl)methyl-3H-
pyrrolo[3,2-d]pyrimidin-4(5H)-one (17). The filtrate (∼206
kg) containing 16 was transferred to a pressure vessel, and 25%
ammonia solution (6.4 kg) was added followed by the addition
of 10% Pd/C (1.0 kg) catalyst suspended in water (10 kg). The
vessel was pressurized to 3 bar with hydrogen gas, and the
contents were stirred at 20-25 °C until there was no more
uptake of hydrogen gas. Upon completion of the reaction (<5%
of 16 remaining by HPLC) the pressure was released from the
vessel which was then flushed several times with nitrogen. The
contents was heated to 45-50 °C and then filtered under
pressure through a lenticular filter. The filtrate was concentrated
by distilling off ∼40% of the solvent under vacuum (1.5 bar)
at 45 °C until 17 precipitated out of the reaction mixture. The
solid was isolated by centrifugation and then dried under
vacuum at 60 °C to furnish the desired product, 17 (25 kg), as
a free base.
4-Chloro-5H-pyrrolo[3,2-d]pyrimidine (14). Compound 2
(500 g, 3.72 mol) was refluxed for 1 h with phosphorous
oxychloride (970 mL, 10.4 mol). The reaction mixture was
cooled to 25-35 °C and poured onto chipped ice (14 L) with
vigorous stirring. The pH of the solution was adjusted to
7.0-8.5 using 30% ammonium hydroxide (∼1.5 L). The
resulting precipitate was collected by vacuum filtration and
washed with water (4 L). The resulting solid was dried in Vacuo
at 100 °C to furnish 14 (551 g, 97%) as a yellow solid. Mp
1
191-193 °C; H NMR (300 MHz, DMSO-d6): δ 12.43 (s, 1
H, D2O exchangeable), 8.61 (s, 1 H), 7.97 (dd, J ) 2.8, 2.8
Hz, 1 H), 6.72 (dd, J ) 1.7, 3.5 Hz, 1 H); 13C NMR (75 MHz,
DMSO-d6): δ 151.3, 149.6, 142.1, 134.8, 124.3, 102.7; IR νmax
3128, 3078, 2979, 1621 cm-1. Elem. anal.: calculated for
C6H4N3Cl: C, 46.93; H, 2.63; N, 27.36; Cl 23.09, found: C,
47.10; H, 2.79; N, 27.15; Cl 22.93.
4-Benzyloxy-5H-pyrrolo[3,2-d]pyrimidine (15). Com-
pound 14 (540 g, 3.51 mol) was refluxed for 48 h with 1 M
solution of sodium benzyloxide in benzyl alcohol (3 L). The
reaction mixture was cooled to 0-5 °C and neutralized to pH
6.5-7.5 with addition of glacial acetic acid (750 mL). Water
(8 L) was added to the reaction mixture and stirred for 2 h
followed by addition of ethyl acetate (10 L). The phases were
split, the aqueous phase was extracted further with ethyl acetate
(3 × 10 L), and the organic layer was evaporated to dryness to
furnish a syrup. The crude was crystallized in hot ethyl acetate
(6 L). The product was filtered and dried overnight to furnish
7-(((4R)-3-Hydroxymethyl)pyrrolidin-1-yl)methyl-3H-
pyrrolo[3,2-d]pyrimidin-4(5H)-one Hydrochloride (1). A
solution of 17 (25 kg, 94.7 mol), water (32 kg), 32%
hydrochloric acid (10.3 kg, 0.95 equiv), and ethanol (224 kg)
were stirred for 30 min and then refluxed at 72-75 °C for 1 h.
The suspension was cooled to 20-25 °C over 1 h and stirred
for an additional 3 h. The product was filtered by centrifugation,
Vol. 13, No. 5, 2009 / Organic Process Research & Development
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931

washed with ethanol (100 kg), and dried in a vacuum oven at
70 °C. The crude was taken directly to the crystallization step.
The crude material and activated charcoal (1.5 kg) in water
(60 kg) were stirred at 60 °C for 30 min and then transferred
via a lenticular filter to another reactor. The crude was further
washed with water (15 kg). The filtrate (∼50 kg of water) was
distilled down at 1.5 bar followed by addition of ethanol (235
kg) to furnish a suspension. Upon complete addition of ethanol,
the contents of the reactor were refluxed for 1 h at 72-75 °C.
The contents of the reactor was cooled to 20-25 °C over 1 h
and stirred for an additional 3 h. The precipitated product was
isolated by centrifugation and then dried under vacuum at 70
°C to constant weight to furnish BCX-4208 (1) as a white solid
(25.3 kg, 85% yield). HPLC analysis: 99.8%. Mp 226 °C; 1H
NMR (300 MHz, DMSO-d6): δ 12.46 (bs, 1 H), 12.14 (bs, 1
H), 10.89 (bs, 1 H), 7.91 (d, J ) 2.6 Hz, 1 H), 7.65 (d, J ) 2.6
Hz, 1 H), 5.49 (s, 1 H), 4.90 (bs, 1 H), 4.33 (m, 2 H), 4.07-4.14
(m, 1 H), 3.04-3.56 (m, 6 H), 2.22 (m, 1 H); 13C NMR (75
MHz, DMSO-d6): δ 153.5, 143.6, 142.6, 129.8, 117.9, 105.9,
70.1, 59.8, 58.6, 53.2, 48.2, 47.1; IR νmax 3339, 3104, 2900,
1706, 1597 cm-1. Elem. anal.: calculated for C12H17ClN4O3:
C, 47.93; H, 5.70; N, 18.63; Cl, 11.79, found: C, 47.70; H,
5.45; N, 18.43; Cl, 11.90.
Received for review April 9, 2009.
OP9001142
932
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Vol. 13, No. 5, 2009 / Organic Process Research & Development