Synthesis of homochiral tetrahydropteridines

Article abstract of DOI:10.1016/j.tet.2014.06.048

A synthesis of protected homochiral tetrahydropteridines from (2S)-malic acid has been developed. This presents methodology for the synthesis of reduced pteridine coenzymes and pharmaceuticals.

Full text of DOI:10.1016/j.tet.2014.06.048

Tetrahedron 70 (2014) 7221e7228
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of homochiral tetrahydropteridines
y
z
*
Stephen J. Baker , Kenneth J.M. Beresford , Douglas W. Young
Department of Chemistry, University of Sussex, Falmer, Brighton, East Sussex BN1 9QJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 April 2014
Received in revised form 11 June 2014
Accepted 13 June 2014
A synthesis of protected homochiral tetrahydropteridines from (2S)-malic acid has been developed. This
presents methodology for the synthesis of reduced pteridine coenzymes and pharmaceuticals.
Ó 2014 Published by Elsevier Ltd.
Available online 18 June 2014
Dedicated to the memory of Professor Sandy
McKillop
Keywords:
Tetrahydropteridines
Stereospecific
Synthesis
Homochiral
1. Introduction
The reduced pteridine coenzyme, tetrahydrofolic acid 1, is im-
portant for mediating enzyme-catalysed one-carbon transfer re-
actions.1 Its involvement in the one-carbon transfer catalysed by
thymidylate synthase (EC 2.1.1.45), which converts deoxyuridine
monophosphate 2 into thymidine monophosphate 3 in a process
requiring the enzyme dihydrofolate reductase (EC 1.5.1.3) for co-
enzyme regeneration, makes it important in the design of anti-
cancer chemotherapeutics. The cancer rescue agent folinic acid 4,
which allows larger doses of the drug methotrexate 5 to be used in
medicine is a one-carbon adduct of tetrahydrofolic acid.
The coenzyme 1 and the related cofactor tetrahydrobiopterin 6,
which mediates enzyme-catalysed aromatic amino acid hydroxyl-
ation are biosynthesised from guanosine triphosphate (GTP) 7 in
several enzyme-catalysed steps by microorganisms. Mammals,
however, cannot synthesise tetrahydrofolate 1 by this route and
require to take the vitamin folic acid 8 in their diet, reducing it to
the coenzyme using dihydrofolate reductase. This makes the en-
zymes involved in the microbiological synthesis targets for anti-
bacterial drugs.
* Corresponding author. E-mail address: d.w.young@sussex.ac.uk (D.W. Young).
y
Present address: GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA,
19426-0989, USA.
z
Present address: Leicester School of Pharmacy, Faculty of Health and Life Sci-
ences, De Montfort University, The Gateway, Leicester, LE1 9BH, UK.
http://dx.doi.org/10.1016/j.tet.2014.06.048
0040-4020/Ó 2014 Published by Elsevier Ltd.

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S.J. Baker et al. / Tetrahedron 70 (2014) 7221e7228
Most homochiral reduced pteridines have so far been accessed by
anhydride with iso-butyl chloroformate and subsequent reaction
with sodium azide gave the corresponding azide. Heating resulted
in Curtius rearrangement and cyclisation of the intermediate iso-
cyanate to give the oxazolidinone 18. Although we had prepared
a useful synthetic intermediate for our target compound 12,
problems were encountered in scaling up and so the alternative
route shown in Scheme 3 was developed.
semi-synthetic or biological methods and we now wish to report
a totally synthetic method, which will allow access to a variety of
targets containing the natural stereochemistry at C-6. Our retro-
synthetic plan, suggested in Scheme 1, requires inversion of stereo-
chemistry in the step 9010 to obtain the appropriate
stereochemistry at C-6, and so (2S)-malic acid 13 was chosen as
starting material. Since we had already developed methods for
obtaining large quantities of samples of (2S)-malic acid, 13, HA¼2H
and 13, HB¼2H, which are deuteriated stereospecifically at C-3,2 the
synthesis will also allow for the preparation of samples of the co-
enzymes 1 and 6, which are stereospecifically labelled at C-7 for
studies of the Amadori rearrangement involved in their biosynthesis.
a
-Methyl (2S)-malate 20, prepared using the method of Miller,4
was reacted with diphenylphosphoryl azide and triethylamine.
Curtius rearrangement of the resultant azide with spontaneous
cyclisation of the intermediate isocyanate gave the oxazolidinone
21 in 74% yield. This was converted into the urethane 22 in 95%
yield using di-tert-butyl dicarbonate, triethylamine and dimethy-
laminopyridine in dioxane and the ester was reduced using sodium
borohydride in tetrahydrofuran at ꢀ15 ꢁC to afford the alcohol 23.
Although this was converted into the tert-butyldiphenylsilyl ether
24 using tert-butyldiphenylsilyl chloride, DMAP and triethylamine
in dichloromethane, various attempts to hydrolyse this directly to
the compound 26 proved fruitless. The alcohol 23, however, un-
derwent cleavage using caesium carbonate in methanol at room
temperature to afford the diol 25 and this was converted into the
silyl ether 26 in 87% yield on reaction with tert-butyldiphenylsilyl
chloride, triethylamine and DMAP in dichloromethane at room
temperature. The urethane protecting group was now removed
using trifluoroacetic acid and the resultant amine 27 was reacted in
triethylamine and methanol with 2-amino-6-chloro-5-nitro-4-
(3H)-pyrimidinone 11, prepared by the method of Wood,5 to give
the adduct 28.
Various unsuccessful attempts were made to convert the product
28 to an amino-mesylate analogue of compound 10 (X¼OMs), which
might be induced to cyclise to the required reduced pteridine. Ar-
guing that the free 6-amino moiety of 28 might form an aziridine
intermediate, and encouraged by the fact that a benzylated analogue
had been shown to cyclise,6 we converted the amine 27 into the
benzyl derivative 29 in 64% yield by reactionwith benzaldehyde and
triethylamine in ethanol followed by in situ reduction with sodium
borohydride as shown in Scheme 4. Reaction with 2-amino-6-
chloro-5-nitro-4-(3H)-pyrimidinone 11 then gave the product 30
in good yield.
The pyrimidine 30 was now converted into the triflate using
triflic anhydride and pyridine and this was hydrogenated in
tetrahydrofuran containing catalytic quantity of 10% palladium on
carbon. The product displayed m/z (FAB, 3-NBA) 526, which was in
keeping with [MþH]þ for the desired compound 31 but the 1H NMR
spectrum was ill-resolved and the product showed a tendency to
oxidise. We therefore repeated the ring-closure reaction but im-
mediately treated the product 31 with freshly prepared7 formic
acetic anhydride. The product was purified by extensive HPLC in
19% yield and had the spectroscopic characteristics of the desired
compound 32 containing a small amount of bis formylated
material.
When the methoxymethylene protected compound 37 was
prepared from the oxazolidinone 21 as outlined in Scheme 5 and
described in the Experimental section, mesylation followed by
hydrogenation using 10% palladium on charcoal in methanol gave
a compound, which was reacted with freshly prepared7 formic
acetic anhydride in pyridine.
O
N
O
N
R
R
HA
HA
H
N
X
NH2
HN
HN
HA
HB
HA
HB
7
H2N
H2N
N
H
N
R'
9
10
O
R
HA
HA
NO2
Cl
HO
HO
CO2H
HN
+
HA
HB
3
HA
HB
H2N
N
HN
R'
HO2C
13
11
Scheme 1.
12
2. Results and discussion
Our first tasks were selectively to convert the
of (2S)-malic acid 13 into a potential side chain and to convert the
-carboxyl group into an amine. Conversion of the -carboxylate
into a hydroxymethyl group has been achieved by Saito et al.3 who
selectively reduced the -ester of diethyl (2S)-malate 14 in good
a-carboxyl group
b
a
a
yield and we used this reaction to obtain the diol 15 in 87% yield as
shown in Scheme 2.
OH
H
HO
CO2Et
H
HO
3
4
5
(i)
4
OH
2
O
H
3
2
3
EtO2C
EtO2C
O
15
14
19
(ii)
OCPh3
OCPh3
OCPh3
4
6
4
HO
3
HO
5
3
O
(iv)
(iii)
H
O
H
H
2
2
4
N
H
HO2C
EtO2C
18
17
16
Scheme 2. Reagents and conditions: (i) Ref. 3; (ii) Ph3CCl, pyridine, rt 1 h, then 50 ꢁC,
4 h (95%); (iii) 1 N aq NaOH, THF, rt, 36 h (93%); (iv) (a) ClCOi2Bu, Et3N, THF, ꢀ30 ꢁC, 1 h,
(b) NaN3, H2O, 0 ꢁC, 1.5 h, (c) toluene, 60 ꢁC, 1 h, then reflux, 30 min (30%).
The spectra of the product indicated that it was the cyclised
triformyltetrahydropteridine 38, which interestingly, appeared to
exist as the 4-phenol tautomer rather than the more usual 3,4-
amide. In NOE experiments, summarised in Fig. 1, irradiation of
This compound spontaneously formed the lactone 19 on
standing and so it was immediately converted into the trityl de-
rivative 16 in 95% yield by reaction with triphenylmethyl chloride in
pyridine. Hydrolysis using aqueous sodium hydroxide in tetrahy-
drofuran gave the acid 17. This was converted into the mixed
the multiplet at
singlet at 8.70 ppm for the 5-formyl proton and of the peaks due
to H-7 and H-9. Irradiation of the broad singlet at 12.1 ppm also
caused enhancement of the singlet at 8.70 ppm for the 5-formyl
proton. Irradiation of the singlet at 8.60 ppm for one 4-N-formyl
d 4.86 ppm for H-6 caused enhancement of the
d
d
d
d

S.J. Baker et al. / Tetrahedron 70 (2014) 7221e7228
7223
Scheme 3. Reagents and conditions: (i) (PhO)2P(O)N3, Et3N, C6H6, reflux, 1 h (74%); (ii) (tBuOCO)2O, Et3N, DMAP, dioxane, rt, 1 h (95%); (iii) NaBH4, THF, ꢀ15 ꢁC, 20 min, (83%); (iv)
tBu(Ph)2SiCl, Et3N, DMAP, CH2Cl2, rt 1 day (65%); (v) Cs2CO3, MeOH, rt, 18 h (86%); (vi) tBu(Ph)2SiCl, Et3N, DMAP, CH2Cl2, rt overnight (87%); (vii) F3CCO2H, 20 min 0 ꢁC (quant); (viii)
11, Et3N, MeOH, 60 ꢁC, then reflux 2 h, then 4 ꢁC, overnight (72%).
4. Experimental
OTBDPS
O
N
OTBDPS
OTBDPS
HO
2'
3'
5
NO2
2
2
3
3
HO
HO
HN
HN
(i)
(ii)
4.1. General
H
H
H
1'
1
1
6
2
H2N
H2N
N
CH2Ph
(iii)
Melting points were determined on a Kofler hot-stage appara-
CH2Ph
29
tus. Optical rotations (given in units of 10ꢀ1 deg cm2 ꢀ1) were
g
27
30
recorded on a PerkineElmer PE241 polarimeter using a 1 dm path-
length micro cell. IR spectra were recorded on a PerkineElmer 1710
Fourier transform instrument and UV spectra on an ATI Unicam
UV2-100 Fourier transform scanning spectrophotometer. 1H NMR
spectra were recorded on Bruker AC-P 250 (250 MHz), DPX 300
(300 MHz), WM 360 (360 MHz) and AMX 500 (500 MHz) Fourier
transform instruments. 13C NMR spectra were recorded on a Bruker
DPX 300 (75.48 MHz) Fourier transform instrument. INEPT and
DEPT experiments were used to help assign 13C resonances where
necessary. Residual undeuteriated solvent peaks were used as in-
ternal references in the NMR spectra. J values are given in Hertz.
Mass spectra were recorded on Kratos MS80RF and Fisons In-
struments VG Auto Spec double focussing spectrometers by Dr. A.
Abdul-Sada. Accurate mass measurements were carried out by the
EPSRC National Mass Spectrometry Service Centre, Swansea. Mi-
croanalyses were performed at Glaxo-Wellcome, Stevenage and by
Medac Ltd. Column chromatography was performed using Merck
Kieselgel 60 (230e400 mesh)dArt 9385 and Fluka Silica gel 60
(220e440 mesh). HPLC was performed using a Waters Symmetry
O
N
O
N
OTBDPS
OTBDPS
CHO
H
N
H
(iv)
N
6
9
HN
HN
H
7
H2N
N
H2N
N
CH2Ph
CH2Ph
32
31
Scheme 4. Reagents and conditions: (i) PhCHO, Et3N, EtOH, reflux, 1 h, NaBH4, rt,
45 min (64%); (ii) 11, Et3N, MeOH, 60 ꢁC, then reflux 4 h, then 4 ꢁC, overnight (76%); (iii)
(a) (CF3CO)2O, pyridine, CH2Cl2, ꢀ30 ꢁC, then 0 ꢁC, 1 h (b) H2, 10% PdeC, THF, rt,
overnight, (iv) HCO2COCH3, pyridine, rt, overnight (19%).
OMOM
6
H
H
H
H
CO2Me
6
CO2Me
CH2OH
(iii)
O
O
O
N
O
N
(i)
(ii)
5
5
5
O
O
O
O
4
4
4
N
H
N
CH2Ph
CH2Ph
CH2Ph
21
35
33
34
C
18, 3.5
m
m, 4.6ꢂ150 mm column (analytical) and a Waters Sym-
(iv)
metry Prep C18, 7
m
m, 19ꢂ150 mm column (preparative) on a Wa-
OMOM
O
N
OH
4
OMOM
3
OMOM
H
CHO
N
ters Millenium HPLC fitted with a Waters 600 controller, Waters
996 Photodiode Array detector, Waters 717 plus autosampler and
Waters in-line degasser. Petroleum ether refers to that fraction of
hexanes bp 60e80 ꢁC.
3'
(vi)
2'
HO
NO2
9
HO
HN
(v)
N
HN
6
2
H
H
6
1
7
1'
2
H2N
N
(OHC)2N
N
N
CH2Ph
CH2Ph
CH2Ph
37
36
38
Scheme 5. Reagents and conditions: (i) (a) LDA, THF, HMPA, ꢀ78 ꢁC, then PhCH2Br,
ꢀ78 ꢁC, 2 h, rt, 2 h (51%); (ii) NaBH4, THF, ꢀ15 ꢁC, 15 min then MeOH (89%); (iii) P2O5,
(MeO)2CH2, CHCl3, rt, 1.5 h (79%); (iv) 2 M aq NaOH, MeOH, reflux, 12 h (90%); (v) 11,
MeOH, Et3N, 60 ꢁC, reflux, 7 h (80%); (vi) (a) MsCl, Et3N, CH2Cl2, 0 ꢁC, 10 min, (b) H2,
MeOH, 10% PdeC, rt, 1.5 h, (c) HCO2COCH3, pyridine, 0 ꢁC, then rt, overnight (33%).
4.2. Ethyl (3S)-3,4-dihydroxybutanoate (15)
This was prepared using the method of Saito et al.3 from diethyl
(2S)-malate 14 in 87% yield as a clear oil; m/z [þve FAB (3-NBA)] 149
([MþH]þ); nmax (film)/cmꢀ1 3399 (br, OH) and 1735 (ester); dH
proton caused enhancement of the singlet at
d 8.88 ppm for the
other 4-N-formyl proton.
3. Conclusions
We have succeeded in developing a synthesis of protected
homochiral tetrahydropteridines with the stereochemistry
of natural coenzymes at C-6 from (2S)-malic acid. This will be of use
in synthesis of reduced pteridine coenzymes and pharmaceuticals.
Fig. 1. NOE experiments on the reduced pteridine 38.

7224
S.J. Baker et al. / Tetrahedron 70 (2014) 7221e7228
(360 MHz, C2HCl3) 1.28 (3H, t, J 7.15, CH3), 2.48 (1H, dd, J2B,2A 16.4,
J2B,3 4.3, H-2B), 2.55 (1H, dd, J2A,2B 16.4, J2A,3 8.3, H-2A), 3.52, (1H,
dd, J4B,4A 11.3, J4B,3 6.2, H-4B), 3.68 (1H, dd, J4A,4B 11.3, J4A,3 3.5, H-
4A), 4.15 (1H, m, H-3) and 4.18 (2H, q, J 7.2, OCH2). This compound
decomposed to give (4S)-4-hydroxydihydrofuran-2(3H)-one 19
upon prolonged exposure to silica gel or storage and so was freshly
prepared as required. (4S)-4-Hydroxydihydrofuran-2(3H)-one 19
H-2B), 2.63 (1H, dd, J2A,2B 16.4, J2A,3 4.6, H-2A), 3.17 (1H, dd, J4B,4A
9.5, J4B,3 5.8, H-4B), 3.25 (1H, dd, J4A,4B 9.5, J4A,3 4.9, H-4A), 4.24 (1H,
m, H-3) and 7.24e7.56 (15H, m, aromatic); dC (75.48 MHz, C2HCl3)
37.0 (C-2), 65.45 (C-4), 66.46 (C-3), 85.74 (OCPh3), 126.1e127.6 (Ar),
142.61 (acid) and 145.78 (Ar).
4.5. (5S)-5-(Triphenylmethyloxymethyl)-oxazolidin-2-one
(18)
was isolated by column chromatography as a clear oil; ½a D26
ꢃ
ꢀ69.2 (c
31
1, MeOH) (lit8
[
a]
ꢀ83.2 (c 0.41, EtOH)); m/z [þve FAB (3-NBA)]
D
103 ([MþH]þ) and 205 ([2MþH]þ); nmax (film)/cmꢀ1 3414 (br, OH)
and 1771 (lactone); dH (300 MHz, C2H3O2H) 2.37 (1H, d, J3B,3A 17.8,
H-3B), 2.83 (1H, dd, J3A,3B 17.8, J3A,4 5.8, H-3A), 4.23 (1H, d, J5B,5A
10.0, H-5B), 4.43 (1H, dd, J5A,5B 10.0, J5A,4 4.2, H-5A) and 4.75 (1H, m,
H-4); dC (75.48 MHz, C2H3O2H) 37.48 (C-3), 67.31 (C-4), 76.72 (C-5)
and 178.12 (lactone).
Triethylamine (1.95 mL, 14 mmol) was added to a solution of
(3S)-3-hydroxy-4-O-triphenylmethyloxybutanoic acid 17 (2.54 g,
7 mmol) in tetrahydrofuran (40 mL) under nitrogen. The mixture
was cooled to ꢀ30 ꢁC and iso-butyl chloroformate (0.94 mL,
7.14 mmol) was added dropwise. A precipitate slowly formed and
the reaction was left for 1 h. The suspension was filtered onto tet-
rahydrofuran (100 mL) at ꢀ30 ꢁC. An ice-cooled solution of sodium
azide (547 mg, 8.4 mmol) in water (25 mL) was added and the
temperature was allowed to warm to 0 ꢁC over 1 h and stirred at
0 ꢁC for a further 30 min. The precipitate was removed by filtration
and the solvents were removed under reduced pressure giving
a white residue. Chloroform (100 mL) was added, forming a sus-
pension, which was washed with water (4ꢂ50 mL) and brine
(50 mL). The chloroform layer was dried (MgSO4) and the solvent
was removed in vacuo to give a foam, which was dissolved in tol-
uene (80 mL) and heated to 60 ꢁC with stirring for 1 h. The reaction
was heated at reflux with stirring for a further 30 min and cooled.
The solvent was removed under reduced pressure to give the crude
product as a foam. Column chromatography on silica gel using ethyl
acetate/petroleum ether (1:1) as eluent gave (5S)-5-(triphenylme-
thyloxymethyl)-oxazolidin-2-one 18 as a white solid (760 mg, 30%);
4.3. Ethyl (3S)-3-hydroxy-4-O-triphenylmethyloxybutanoate
(16)
Triphenylmethyl chloride (752 mg, 2.69 mmol), dried under
high vacuum and stored overnight in a vacuum desiccator, was
added to
a solution of freshly prepared ethyl (3S)-3,4-
dihydroxybutanoate 15 (200 mg, 1.35 mmol) in dry pyridine
(2 mL) under nitrogen. The resulting yellow solution was stirred at
room temperature for 1 h and heated at 50 ꢁC with stirring for
a further 1 h. Pyridine (1 mL) was added and the reaction was
stirred at 50 ꢁC for 4 h. Ethyl acetate (20 mL) was added and the
mixture was washed with water (2ꢂ10 mL) and brine (10 mL). The
organic layer was dried (MgSO4) and the solvent was removed in
vacuo. The residue was azeotroped with toluene (2ꢂ10 mL) to give
a yellow crystalline solid. Column chromatography on silica gel
using ethyl acetate/petroleum ether (1:4) as eluent gave ethyl (3S)-
3-hydroxy-4-O-triphenylmethyloxybutanoate 16 as a yellow oil,
mp 198e200 ꢁC; ½a D26
þ13.9 (c 0.85, CHCl3); (found: C, 76.65; H, 5.9;
ꢃ
N, 3.7, C23H21NO3 requires: C, 76.9; H, 5.9; N, 3.9%); m/z [þve FAB (3-
NBA)] 360 ([MþH]þ) and 382 ([MþNa]þ); nmax (KBr)/cmꢀ1 3415 (br,
NH), 1772 and 1720 (urethane and amide); dH (360 MHz, C2HCl3)
3.24 (1H, dd, J4B,4A 10.4, J4B,5 4.5, H-4B), 3.40 (1H, dd, J4A,4B 10.4, J4A,5
4.3, H-4A), 3.46 (1H, m, H-6B), 3.62 (1H, m, H-6A), 4.75 (1H, m, H-
5), 5.18 (1H, s, NH, exch. with 2H2O) and 7.23e7.56 (15H, m, ArH); dC
(75.48 MHz, C2HCl3) 41.56 (C-4), 63.12 (C-6), 74.38 (C-5), 85.80
(OCPh3), 126.2e142.37 (Ar) and 157 (urethane).
which crystallised on standing (501 mg, 95%); mp 92e94.5 ꢁC; ½a D26
ꢃ
ꢀ5.3 (c 1, CHCl3); (found: C, 77.0; H, 6.8, C25H26O4 requires C, 76.9;
H, 6.7%); m/z [þve FAB (3-NBA)] 413 ([MþNa]þ); nmax (film)/cmꢀ1
3438 (br, OH) and 1730 (ester); dH (300 MHz, C2HCl3) 1.30 (3H, t, J
7.1, CH3), 2.57 (1H, dd, J2B,2A 16.1, J2B,3 7.7, H-2B), 2.64 (1H, dd, J2A,2B
16.1, J2A,3 4.6, H-2A), 3.08 (1H, br s, OH, exch. with 2H2O), 3.23 (2H,
d, J4,3 5.4, H-4), 4.20 (2H, q, J 7.1, OCH2), 4.29 (1H, m, H-3) and
7.24e7.77 (15H, m, ArH); dC (75.48 MHz, C2HCl3) 16.13 (CH3), 40.51
(C-2), 62.69 (C-4), 68.52 (OCH2), 69.58 (C-3), 88.69 (OCPh3),
129.1e145.71 (Ar) and 174.28 (ester).
4.6. Methyl (5S)-oxazolidin-2-one-5-carboxylate (21)
Triethylamine (7.05 mL, 51 mmol) was added to a suspension of
a
-methyl (2S)-malate 204 (5.0 g, 34 mmol) in benzene (180 mL)
4.4. (3S)-3-Hydroxy-4-O-triphenylmethyloxybutanoic acid
(17)
under nitrogen at room temperature, dissolving the suspension.
Diphenylphosphoryl azide (8.32 mL, 38 mmol) was added dropwise
and the resulting mixture was heated at reflux with stirring for 1 h
and cooled to room temperature. The solvents were removed in
vacuo to yield the crude product as a red oil. Column chromatog-
raphy on silica gel using ethyl acetate/petroleum ether (2:1) as el-
uent gave methyl (5S)-oxazolidin-2-one-5-carboxylate 21 as
a yellow solid (3.62 g, 74%). A small sample was recrystallised from
1 N Aqueous sodium hydroxide (2 mL) was added to a solution
of ethyl (3S)-3-hydroxy-4-O-triphenylmethyloxybutanoate 16
(464 mg, 1.2 mmol) in tetrahydrofuran (15 mL) and the mixture was
stirred at room temperature for 36 h. The sodium salt of the
product, (3S)-3-hydroxy-4-O-triphenylmethyloxybutanoic acid 17,
was extracted into water (2ꢂ15 mL) and the water layer was
washed with ethyl acetate (2ꢂ10 mL). Ethyl acetate (30 mL) was
added to the water layer and compressed air was passed through
the mixture to ensure thorough mixing of the layers. 1 N Aqueous
hydrochloric acid was added dropwise to the mixture until the pH
was 4e5. A white precipitate formed and dissolved in the ethyl
acetate. The layers were separated and the ethyl acetate layer was
washed with water (2ꢂ15 mL) and brine (10 mL) and dried
(MgSO4). The solvent was removed in vacuo to yield (3S)-3-
hydroxy-4-O-triphenylmethyloxybutanoic acid 17 as a clear glass
ethyl acetate; mp 98e99 ꢁC; ½a D26
ꢀ15.7 (c 1.0, MeOH); m/z [þve
ꢃ
FAB (3-NBA)] 146 ([MþH]þ), 291 ([2MþH]þ), 436 ([3MþH]þ) and
581 ([4MþH]þ); nmax (KBr)/cmꢀ1 3277 (NH), 1763 (ester) and 1736
(urethane); dH (300 MHz, C2HCl3) 3.64 (1H, dd, J4B,4A 9.3, J4B,5 5.5, H-
4B), 3.78 (3H, s, OCH3), 3.84 (1H, t, J4A,4B¼J4A,5¼9.3, H-4A), 4.98 (1H,
dd, J5,4A 9.3, J5,4B 5.5, H-5) and 6.26 (1H, br s, NH, exch. with
MeO2H); dC (75.48 MHz, C2HCl3) 45.43 (C-4), 54.78 (OCH3), 74.37
(C-5), 160.83 (urethane) and 171.18 (ester).
4.7. Methyl (5S)-3-tert-butoxycarbonyloxazolidin-2-one-5-
carboxylate (22)
(398 mg, 93%); mp 85e89 ꢁC; ½a D26
ꢃ
ꢀ8.3 (c 1.0, CHCl3); (found C,
74.1; H, 5.9, C23H22O4$0.5H2O requires C, 74.4; H, 6.2%); m/z [þve
FAB (3-NBA)] 385 ([MþNa]þ); nmax (KBr)/cmꢀ1 3431 (br, OH) and
1709 (acid); dH (360 MHz, C2HCl3) 2.56 (1H, dd, J2B,2A 16.4, J2B,3 7.8,
A solution of di-tert-butyl dicarbonate (7.14 g, 33 mmol) in 1,4-
dioxane (8 mL) was added to a solution of methyl (5S)-oxazolidin-

S.J. Baker et al. / Tetrahedron 70 (2014) 7221e7228
7225
2-one-5-carboxylate 21 (3.17 g, 22 mmol) in 1,4-dioxane (80 mL),
triethylamine (4.56 mL, 33 mmol) and dimethylaminopyridine
(533 mg, 4.3 mmol) under nitrogen. The reaction was stirred at
room temperature for 1 h with evolution of CO2. The solvents were
removed in vacuo and the residue was dissolved in ethyl acetate
(75 mL). The ethyl acetate was washed with water (2ꢂ50 mL) and
brine (30 mL), and dried (MgSO4). The solvent was removed in
vacuo to yield the product as a red oil, which crystallised on
standing (5.09 g, 95%). A small sample was purified further by
column chromatography on silica gel using ethyl acetate/petroleum
ether (2:3) as eluent to yield an analytical sample of methyl (5S)-3-
tert-butoxycarbonyloxazolidin-2-one-5-carboxylate 22 as an or-
acetate/petroleum ether (1:4) as eluent afforded (5S)-3-tert-
butoxycarbonyl-5-(O-tert-butyldiphenylsilyloxymethyl)-oxazoli-
din-2-one 24 as a clear oil, which crystallised on standing (1.13 g,
65%); mp 80e83 ꢁC; ½a D26
þ31.4 (c 1.0, CHCl3); m/z (FAB, PEGNa/
ꢃ
NOBA) found 478.2009, [C25H33NO5SiþNa] requires 478.2026; m/z
[þve FAB (3-NBA)] 456 ([MþH]þ) and 478 ([MþNa]þ); nmax (KBr)/
cmꢀ1 1805 and 1722 (urethane and imide); dH (300 MHz, C2HCl3)
0.96 (9H, s, SiC(CH3)3), 1.48 (9H, s, OC(CH3)3), 3.59 (1H, dd, J4B,4A
11.5, J4B,5 2.8, H-4B), 3.81 (1H, dd, J4A,4B 11.5, J4A,5 3.1, H-4A), 3.87
(2H, d, J6,5 7.0, H-6), 4.46 (1H, m, H-5) and 7.28e7.61 (10H, m, ArH);
dC (75.48 MHz, C2HCl3) 19.59 (SiC(CH3)3), 27.09 (SiC(CH3)3), 28.41
(OC(CH3)3), 45.43 (C-4), 64.65 (C-6), 72.81 (C-5), 84.03 (OC(CH3)3),
128.26e136.05 (Ar), 149.97 and 152.32 (urethane).
ange oil, which crystallised on standing; mp 75e76 ꢁC; ½a D26
þ24.5
ꢃ
(c 1.0, CHCl3); (found C, 49.2; H, 6.2; N, 5.7, C10H15NO6 requires C,
49.0; H, 6.2; N, 5.7%); m/z [þve FAB (3-NBA)] 246 ([MþH]þ) and 268
([MþNa]þ); nmax (KBr)/cmꢀ1 1807 (bisurethane), 1755 (ester) and
1719 (imide); dH (300 MHz, C2HCl3) 1.51 (9H, s, C(CH3)3), 3.86 (3H, s,
OCH3), 4.03 (1H, dd, J4B,4A 10.7, J4B,5 5.3, H-4B)), 4.18 (1H, dd, J4A,4B
10.1, J4A,5 9.6 H-4A)) and 4.92 (1H, dd, J5,4A 9.6, J5,4B 5.3, H-5)); dC
(75.48 MHz, C2HCl3) 28.33 (C(CH3)3), 46.53 (C-4), 53.67 (OCH3),
69.30 (C-5), 84.92 (OC(CH3)3), 149.26 and 150.93 (urethane) and
168.96 (ester).
4.10. (2S)-N-tert-Butoxycarbonyl-2,3-dihydroxypropylamine
(25)
Caesium carbonate (901 mg, 2.8 mmol) was added to a solution
of (5S)-3-tert-butoxycarbonyl-5-hydroxymethyloxazolidin-2-one
23 (2.73 g, 12.5 mmol) in methanol (150 mL) under nitrogen and
the mixture was stirred at room temperature for 18 h. The solution
was neutralised by addition of 5% aqueous citric acid (ca. 8 mL). The
methanol was removed under reduced pressure and the residue
was dissolved in ethyl acetate (50 mL). The ethyl acetate was
washed with brine (3ꢂ30 mL) and dried (MgSO4). The solvent was
removed in vacuo to yield (2S)-N-tert-butoxycarbonyl-2,3-
4.8. (5S)-3-tert-Butoxycarbonyl-5-hydroxymethyloxazolidin-
2-one (23)
A solution of methyl (5S)-3-tert-butoxycarbonyloxazolidin-2-
one-5-carboxylate 22 (5.0 g, 20.3 mmol) in tetrahydrofuran
(100 mL) was cooled in an ice/salt water bath to ꢀ15 ꢁC under ni-
trogen and stirred for 15 min. Sodium borohydride (1.16 g,
30.7 mmol) was added in one portion and the mixture was stirred
at ꢀ15 ꢁC for 20 min. Methanol (40 mL) was added causing effer-
vescence. The solution was allowed to warm to room temperature
with stirring over 1 h. The solvents were removed in vacuo and the
residue was partitioned between ethyl acetate (75 mL) and brine
(50 mL). The brine layer was extracted with ethyl acetate (25 mL)
and the combined ethyl acetate layers were washed with brine
(25 mL). The ethyl acetate was removed under reduced pressure to
afford (5S)-3-tert-butoxycarbonyl-5-hydroxymethyloxazolidin-2-
one 23 as a red oil, which crystallised on standing (3.66 g, 83%). A
sample was purified by column chromatography on silica gel using
ethyl acetate/petroleum ether (3:1 then 1:1) to yield a white solid;
dihydroxypropylamine 25 as a yellow oil (2.06 g, 86%); ½a D27
þ8.9
ꢃ
(c 1.0, CHCl3); m/z [þve FAB (3-NBA)] 192 ([MþH]þ), 214
([MþNa]þ), 383 ([2MþH]þ) and 405 ([2MþNa]þ); nmax (film)/cmꢀ1
3332 (br, OH, NH) and 1688 (urethane); dH (300 MHz, C2HCl3) 1.37
(9H, s, C(CH3)3), 2.63 (2H, br s, OH, exch. with 2H2O), 3.17 (1H, dd,
J1B,1A 14.4, J1B,2 5.5, H-1B), 3.22 (1H, dd, J1A,1B 14.4, J1A,2 5.1, H-1A),
3.50 (1H, dd, J3B,3A 11.6, J3B,2 5.0, H-3B), 3.55 (1H, dd, J3A,3B 11.6, J3A,2
4.3, H-3A), 3.68 (1H, m, H-2) and 4.89 (1H, br s, NH, exch. with
2H2O); dC (75.48 MHz, C2HCl3) 28.77 (C(CH3)3), 43.16 (C-1), 64.03
(C-3), 71.64 (C-2), 80.36 (OC(CH3)3) and 157.72 (urethane).
4.11. (2S)-N-tert-Butoxycarbonyl-3-(O-tert-butyldiphenylsily-
loxy)-2-hydroxypropylamine (26)
Triethylamine (3.2 mL, 23 mmol) and dimethylaminopyridine
(101 mg, 0.8 mmol) were added to an ice-cold solution of (2S)-N-
mp 83e85 ꢁC; ½a 2D6
ꢃ
þ44.6 (c 1.0, CHCl3); (found C, 49.6; H, 7.2; N,
6.3, C9H15NO5 requires C, 49.8; H, 7.0, N 6.45%); m/z [þve FAB (3-
NBA)] 218 ([MþH]þ) and 240 ([MþNa]þ); nmax (KBr)/cmꢀ1 3449
(OH), 1787 and 1717 (urethane); dH (300 MHz, C2HCl3) 1.47 (9H, s,
C(CH3)3), 2.68 (1H, br s, OH, exch. with MeO2H), 3.62 (1H, dd, J4B,4A
12.7, J4B,5 3.9, H-4B), 3.63 (3H, m, H-4A, H-6A and H-6B) and 4.54
(1H, m, H-5); dC (75.48 MHz, C2HCl3) 28.36 (C(CH3)3), 45.11 (C-4),
62.90 (C-6), 73.50 (C-5), 84.39 (OC(CH3)3), 149.79 and 152.77
(urethane).
tert-butoxycarbonyl-2,3-dihydroxypropylamine
25
(1.99
g,
10.3 mmol) in dichloromethane (30 mL) under a nitrogen atmo-
sphere. tert-Butyldiphenylsilyl chloride (2.97 mL, 11.4 mmol) was
added dropwise to the mixture and the solution was stirred over-
night. The solution became yellow and a precipitate formed. The
organic solution was washed with water (2ꢂ30 mL) and brine
(20 mL) and dried (MgSO4). The solvent was removed in vacuo to
yield the crude product as a yellow oil. Column chromatography
on silica gel using ethyl acetate/petroleum ether (1:3) as eluent
gave (2S)-N-tert-butoxycarbonyl-3-(O-tert-butyldiphenylsilyloxy)-
2-hydroxypropylamine 26 as a very pale yellow oil (3.86 g, 87%);
4.9. (5S)-3-tert-Butoxycarbonyl-5-(O-tert-butyldiphenylsily-
loxymethyl)-oxazolidin-2-one (24)
a 26
½ ꢃD ꢀ9.0 (c 1.0, CHCl3); m/z (FAB, PEGH/NOBA) found 429.233654,
A
solution of (5S)-3-tert-butoxycarbonyl-5-hydroxymethyl
C24H35NO4Si
requires
429.233537;
found
430.238639,
oxazolidin-2-one 23 (820 mg, 3.8 mmol) in dichloromethane
(10 mL) was cooled in an ice/water bath under a nitrogen atmo-
sphere. Triethylamine (0.58 mL, 4.1 mmol) was added followed by
dimethylaminopyridine (18 mg, 0.15 mmol) and tert-butyldiphe-
nylsilyl chloride (1.08 mL, 4.1 mmol). The mixture was stirred at
room temperature for 1 day and the solvent was removed under
reduced pressure. The residue was dissolved in ethyl acetate
(20 mL) and washed with water (2ꢂ15 mL) and brine (10 mL), and
dried (MgSO4). The solvent was removed in vacuo to yield the crude
product as an oil. Column chromatography on silica gel using ethyl
[C24H35NO4SiþH]þ requires 430.241362; nmax (film)/cmꢀ1 3428 (br,
OH, NH) and 1694 (urethane); dH (300 MHz, C2HCl3) 0.98 (9H, s,
SiC(CH3)3), 1.34 (9H, s, OC(CH3)3), 2.80 (1H, br s, OH, exch. with
2H2O), 3.05 (1H, dd, J1B,1A 14.0, J1B,2 6.7, H-1B), 3.27 (1H, dd, J1A,1B
14.0, J1A,2 3.3, H-1A), 3.52 (1H, dd, J3B,3A 10.3, J3B,2 6.1, H-3B), 3.59
(1H, dd, J3A,3B 10.3, J3A,2 4.8, H-3A), 3.71 (1H, m, H-2), 4.80 (1H, br s,
NH, exch. with 2H2O) and 7.26e7.60 (10H, ArH); dC (75.48 MHz,
C2HCl3) 19.65 (SiC(CH3)3), 27.29 (SiC(CH3)3), 28.78 (C(CH3)3), 43.5
(C-1), 65.89 (C-3), 71.73 (C-2), 79.99 (OC(CH3)3), 128.27e135.95 (Ar)
and 157.0 (urethane).

7226
S.J. Baker et al. / Tetrahedron 70 (2014) 7221e7228
4.12. (2S)-3-(O-tert-Butyldiphenylsilyloxy)-2-hydroxypropyl
amine (27)
ethanol (8 mL) under nitrogen until the pH of the solution was 8.
Benzaldehyde (310 L, 3.0 mmol) was added and the pH was
m
readjusted to 8e9 using triethylamine. The mixture was heated at
reflux for 1 h, allowed to cool to room temperature and further
cooled in an ice/water bath. Sodium borohydride (324 mg,
8.6 mmol) was added very slowly and the reaction was allowed to
warm to room temperature and stirred until effervescence stopped
(about 45 min). The solvents were removed in vacuo and the res-
idue was dissolved in dichloromethane (15 mL). The solution was
washed with water (2ꢂ10 mL) and brine (10 mL) and dried
(MgSO4). The solvent was removed in vacuo to yield the crude
product as a waxy solid. Column chromatography on silica gel using
ethyl acetate/petroleum ether (1:1 then 2:1) as eluent gave (2S)-N-
benzyl-3-(O-tert-butyldiphenylsilyloxy)-2-hydroxypropylamine 29
Trifluoroacetic acid (90 mL) was cooled in an ice/water bath
under a nitrogen atmosphere and added to (2S)-N-tert-butox-
ycarbonyl-3-(O-tert-butyldiphenylsilyloxy)-2-hydroxypropylamine
26 (2.18 g, 5.08 mmol). The solution was stirred for 20 min at 0 ꢁC
and the solvent was removed in vacuo to yield the trifluoroacetate of
(2S)-3-(O-tert-butyldiphenylsilyloxy)-2-hydroxypropylamine 27 as
a pale green solid that was wet with trifluoroacetic acid. This was
azeotroped with ethyl acetate (50 mL) and diethyl ether (50 mL). The
crystalline solid was placed under high vacuum but the final solid
still contained trifluoroacetic acid (2.54 g). A small sample was
treated with triethylamine and extracted into ethyl acetate. The
ethyl acetate was washed with brine, dried (MgSO4) and the solvent
was removed in vacuo to afford an analytical sample of (2S)-3-(O-
tert-butyldiphenylsilyloxy)-2-hydroxypropylamine 27 as a clear oil,
as a white solid (806 mg, 64%); mp 88.5e90 ꢁC; ½a D26
ꢀ6.8 (c 1.0,
ꢃ
CHCl3); (found: C, 74.4; H, 8.0; N, 3.3, C26H33NO2Si requires: C, 74.4;
H, 7.9; N, 3.3%); m/z [þve FAB (3-NBA)] 420 ([MþH]þ) and 362 ([M-
C(CH3)3]þ); nmax (KBr)/cmꢀ1 3422 and 3324 (OH and NH); dH
(250 MHz, C26H6) 1.12 (9H, s, SiC(CH3)3), 2.50 (1H, dd, J1B,1A 11.9, J1B,2
6.5, H-1B), 2.57 (1H, dd, J1A,1B 11.9, J1A,2 4.3, H-1A), 3.45 (1H, d, JB,A
13.3, NCH2Ph), 3.52 (1H, d, JA,B 13.3, NCH2Ph), 3.67 (1H, d, J3B,2 1.4,
H-3B), 3.69 (1H, s, H-3A), 3.74 (1H, m, H-2), 7.04e7.26 and
7.71e7.78 (15H, m, ArH); dC (75.48 MHz, C26H6) 19.64 (SiC(CH3)3),
27.24 (SiC(CH3)3), 51.85 (C-1), 54.20 (NCH2Ph), 67.23 (C-3), 70.79
(C-2) and 127.27e140.99 (Ar).
free from trifluoroacetic acid; ½a D26
ꢀ5.4 (c 1.0, CHCl3); m/z (FAB,
ꢃ
PEGH/NOBA) found 330.187426, [C19H27NO2SiþH]þ requires
330.188933; m/z [þve FAB (3-NBA)] 330 ([MþH]þ); nmax (film)/cmꢀ1
3363 (br, OH, NH); dH (300 MHz, C2HCl3) 0.95 (9H, s, SiC(CH3)3), 2.68
(2H, m, H-1), 3.48 (2H, d, J3,2 5.2, H-3), 3.63 (1H, br s, H-2), 4.07 (3H,
br, NH and OH, exch. with 2H2O), 7.25e7.64 (10H, m, ArH); dC
(75.48 MHz, C2HCl3) 17.34 (SiC(CH3)3), 24.97 (SiC(CH3)3), 43.4 (C-1),
63.94 (C-3), 69.3 (C-2) and 125.76e133.94 (Ar).
4.13. 2-Amino-6-((20S)-30-O-tert-butyldiphenylsilyloxy-20-hy-
droxypropylamino)-5-nitro-4-(3H)-pyrimidinone (28)
4.15. 2-Amino-6-((20S)-N-benzyl-30-O-tert-butyldiphenylsily-
loxy-20-hydroxypropylamino)-5-nitro-4-(3H)-pyrimidinone
(30)
Triethylamine was added to a solution of freshly prepared5 2-
amino-6-chloro-5-nitro-4-(3H)-pyrimidinone
11
(870
mg,
Triethylamine was added to a solution of freshly prepared5 2-
4.5 mmol) in methanol (82 mL) at 60 ꢁC under nitrogen until the pH
was 9e10. Triethylamine was also added to a solution of (2S)-3-(O-
tert-butyldiphenylsilyloxy)-2-hydroxypropylamine 27 (2.025 g,
6.15 mmol) in methanol (5 mL) until the pH was 7. This solution was
added to the hot pyrimidine solution. The pH of the resulting
mixture was adjusted to 9e10 using triethylamine and the mixture
was heated at reflux with stirring for 2 h and cooled to room
temperature. The solvents were removed in vacuo until about
a third of the original volume remained. The resulting suspension
was allowed to stand at 4 ꢁC overnight. The product was collected
by filtration and washed with cold diethyl ether. The product
was dried in a vacuum dessicator to yield 2-amino-6-((20S)-30-O-
tert-butyldiphenylsilyloxy-20-hydroxypropylamino)-5-nitro-4-
(3H)-pyrimidinone 28 as a yellow solid (1.43 g, 66%). A second crop
amino-6-chloro-5-nitro-4-(3H)-pyrimidinone
11
(343
mg,
1.8 mmol) in methanol (40 mL) at 60 ꢁC under nitrogen until the pH
was 9e10. A solution of N-benzyl-3-O-tert-butyldiphenylsilyloxy-
(2S)-hydroxypropanolamine 29 (755 mg, 1.8 mmol) in methanol
(6 mL) was added, the pH of the mixture being 9e10. The solution
was heated at reflux with stirring for 4 h. The reaction was cooled to
room temperature and the solvents were removed in vacuo to yield
the crude product as a yellow foam (1.27 g). This was combined
with the crude product from a further reaction (200 mg). Column
chromatography on silica gel using a gradient solvent system from
ethyl acetate/petroleum ether (2:1) to ethanol/ethyl acetate (3:7) as
eluent afforded 2-amino-6-((20S)-N-benzyl-30-O-tert-butyldiphe-
nylsilyloxy-20-hydroxypropylamino)-5-nitro-4-(3H)-pyrimidinone
30 as a yellow foam (790 mg, 76% total). A second purification on
the mixed fraction afforded a second crop (40 mg); 86.2e87.3 ꢁC;
of product was collected (132 mg, 6%); mp 255e261 ꢁC; [
a
]
D
29 þ26.0
(c 0.75, CHCl3); (found: C, 56.1; H, 6.2; N,14.3, C23H29N5O5Si$0.5H2O
requires; C, 56.1; H, 6.1; N, 14.2%); m/z [þve FAB (3-NBA)] 484
([MþH]þ); nmax (KBr)/cmꢀ1 3308 (br, OH, NH) and 1685 (amide);
lmax (MeOH)/nm 212 and 236 (shoulder), 283 and 332; lmax (OHe,
MeOH) 209 and 344; dH (500 MHz, C2HCl3) 1.11 (9H, s, SiC(CH3)3),
2.77 (overlap with water, br s, OH), 3.52 (1H, m, H-10B), 3.75 (2H, d,
½
a 2D6
ꢃ
þ2.2 (c 1.0, CHCl3); m/z (FAB, PEGH/NOBA) found 574.250170,
[C30H35N5O5SiþH]þ requires 574.248573; nmax (KBr)/cmꢀ1 3438 (br,
OH, NH) and 1673 (amide); lmax (MeOH)/nm 218, 254, 283 and 358
(ε 38,439, 17,509, 7228 and 4590); lmax (OHꢀ) 216, 251 and 366; dH
(300 MHz, [2H6]-DMSO) 0.77 (9H, s, SiC(CH3)3), 2.95 (1H, dd, J1 B,1 A
0
0
13.6, J1 B,2 8.1, H-10B), 3.25e3.42 (3H, m, H-10A, H-30), 3.76 (1H, m,
0
0
J3 ,2 5.2, H-30), 3.91 (1H, m, H-10A), 4.03 (1H, s, H-20), 5.29 (1H, br,
NH), 7.38e7.69 (10H, m, ArH), 8.24 (1H, br s, 2-NH2), 9.76 (1H, t,
H-20), 4.62 (1H, d, JB,A 15.8, NCH2Ph), 4.84 (1H, d, JA,B 15.8, NCH2Ph),
0
0
4.89 (1H, d, JOH,2 5.7, 20-OH, exch. with 2H2O), 7.10e7.50 (15H, m,
0
J6NH,1 5.0, 6-NH) and 10.63 (1H, br s, NH); in a saturation transfer
ArH) and 10.56 (1H, br s, NH, exch. with 2H2O); dC (75.48 MHz,
C2HCl3) 21.43 (SiC(CH3)3), 29.05 (SiC(CH3)3), 54.4 (C-10), 63.6
(NCH2Ph), 67.81 (C-30), 71.7 (C-20), 115.36 (C-5), 130.01e138.00 (Ar),
154.99 (C-2), 161.22 (C-4) and 164.6 (C-6).
0
experiment irradiation of 2-NH at d 8.24 ppm affected the signal at
d
5.29 ppm thus allowing us to define the signals due to the 2-NH2
group; dC (75.48 MHz, C2HCl3) 18.69 (SiC(CH3)3), 26.27 (SiC(CH3)3),
44.03 (C-10), 64.79 (C-30), 73.10 (C-20), 125.25 (C-5), 127.09e134.96
(Ar), 153.20 (C-2), 157.69 (C-4) and 159.18 (C-6).
4.16. (6R)-(2-Amino-8-benzyl-5-formyl-4(3H)-oxo-6-(O-tert-
butyldiphenylsilyloxymethyl)-5,6,7,8-tetrahydropteridine (32)
4.14. (2S)-N-Benzyl-3-(O-tert-butyldiphenylsilyloxy)-2-
hydroxypropylamine (29)
Pyridine (42 mL, 520 mmol) was added to a solution of 2-amino-
6-((20S)-N-benzyl-30-O-tert-butyldiphenylsilyloxy-2-
Triethylamine was added to a solution of (2S)-3-(O-tert-butyl-
diphenylsilyloxy)-2-hydroxypropylamine 27 (1.23 g, 3.7 mmol) in
hydroxypropylamino)-5-nitro-4-(3H)-pyrimidinone 30 (100 mg,
174 mmol) in dichloromethane (1.25 mL) under argon. The resulting

S.J. Baker et al. / Tetrahedron 70 (2014) 7221e7228
7227
solution was cooled to ꢀ30 ꢁC and trifluoromethanesulfonic an-
a yellow oil; ½a 2D3
ꢃ
þ11.6 (c 1, CHCl3); m/z (ammonia CI) found
hydride (38
mL, 227
m
mol) was added. The solution was allowed to
236.0925, [C12H13NO4þH]þ requires 236.0923; m/z [FAB, 3-NBA]
236 ([MþH]þ); nmax (film)/cmꢀ1 1746 (br, C]O); dH (300 MHz,
C2HCl3) 3.49 (1H, dd, J4A,4B 9.4, J4A,5 5.3, H-4A), 3.68 (1H, t,
J4B,4A¼J4B,5¼9.4, H-4B), 3.81 (3H, s, OCH3), 4.44 (2H, AB, JAB 14.9,
NCH2Ph), 4.90 (1H, dd, J5,4A 5.3, J5,4B 9.4, H-5) and 7.25e7.40 (5H, m,
ArH); dC (75.48 MHz, C2HCl3) 47.7 (C-4), 49.3 (NCH2Ph), 54.0
(OCH3), 70.7 (C-5), 129.1, 130.0 and 136.1 (Ar), 157.7 (urethane) and
170.3 (ester).
warm to 0 ꢁC over 1 h. Tetrahydrofuran (25 mL) was added to the
reaction and the flask was evacuated and refilled with argon several
times. 10% Palladium on carbon (500 mg) was added and the
mixture was stirred overnight under hydrogen at room tempera-
ture and pressure. The UV spectrum of the crude reaction mixture
showed lmax 205 and 293 (very small). The solvents were removed
in vacuo and the flask was refilled with argon. Pyridine (0.8 mL) was
added dropwise to the resulting residue under an argon atmo-
sphere. This was cooled in an ice/water bath and freshly prepared7
cold formic acetic anhydride (10 mL) was added dropwise. The
mixture was allowed to warm to room temperature. Effervescence
continued for 3 h. The reaction was stirred overnight at room
temperature. The solvents were removed in vacuo. Dichloro-
methane (10 mL) was added and the solution was filtered. The fil-
trate was washed with water (2ꢂ10 mL) and brine (5 mL) and dried
(MgSO4). The solvent was removed in vacuo to yield the crude
product as an orange oil (70 mg). HPLC on a C18 preparative column
(19 mmꢂ150 mm) using 44% aqueous ammonium formate (0.1%,
pH 7.3) in acetonitrile as eluent at a flow rate of 5 mL minꢀ1 gave
samples, which eluted at 26 min, lmax 200 and 252 nm, and 42 min,
lmax 200 and 257 nm. These were separately azeotroped with
dichloromethane and placed under high vacuum (0.1 mmHg) with
mild heating to remove the ammonium formate by sublimation.
This gave two samples of (6R)-(2-amino-8-benzyl-5-formyl-4(3H)-
oxo-6-(O-tert-butyldiphenylsilyloxymethyl)-5,6,7,8-
4.18. (5S)-3-Benzyl-5-hydroxymethyloxazolidin-2-one (34)
Sodium borohydride (423 mg, 11.18 mmol) was added to a so-
lution of methyl (5S)-3-benzyloxazolidin-2-one-5-carboxylate 33
(1.766 g, 7.52 mmol) in tetrahydrofuran (37 mL) at ꢀ15 ꢁC. After
15 min methanol (15 mL) was added and the solution was allowed
to warm to room temperature. When TLC indicated that no starting
material remained, the solvent was removed in vacuo. The residue
was dissolved in ethyl acetate (30 mL) and washed with saturated
brine (10 mL). The organic layer was dried (MgSO4) and the solvent
was removed in vacuo. Flash chromatography on silica gel using
ethyl acetate/petroleum ether (4:1) as eluent afforded (5S)-3-
benzyl-5-hydroxymethyloxazolidin-2-one 34 (1.386 g, 89%) as
a white solid; mp 70e71 ꢁC; ½a D23
þ17.9 (c 1.06, CHCl3); (found C,
ꢃ
63.5; H, 6.3; N, 6.7. C11H13NO3 requires C, 63.8; H, 6.3; N, 6.8%); m/z
[FAB, 3-NBA] 208 ([MþH]þ); nmax (KBr)/cmꢀ1 3338 (OH) and 1723
(br, OCON); dH (300 MHz, C2HCl3) 2.20e2.36 (1H, br s, OH), 3.34
(2H, ABX, J4A,4B 9.6, J4A,5 7.2, J4B,5 8.4, H-4), 3.70 (2H, ABX, J6A,6B 9.6,
J6A,5 4.8, J6B,5 2.4 H-6), 4.36 (2H, AB, JAB 13.2, NCH2Ph), 4.52 (1H,
dddd, J5,4A 7.2, J5,4B 8.4, J5,6A 4.8, J5,6B 2.4, H-5) and 7.16e7.36 (5H, m,
ArH); dC (75.48 MHz, C2HCl3) 45.5 (C-4), 48.7 (NCH2Ph), 63.4 (C-6),
74.1 (C-5), 128.4, 129.3 and 135.9 (Ar) and 158.5 (C-2).
tetrahydropteridine 32 (10 mg and 9 mg, respectively, 19% total) as
oils; ½a 2D6
þ103.2 (c 0.68, CHCl3); m/z (FAB, PEGH/NOBA) found
ꢃ
554.257376, [C31H35N5O3SiþH]þ requires 554.258744; m/z [þve
FAB (3-NBA)] 1107 ([2MþH]þ), 576 (MþNa]þ) and 554 ([MþH]þ),
with ions for a diformyl by-product at m/z 582 ([MþH]þ) and 604
([MþNa]þ); nmax (thin film)/cmꢀ1 3269 (NH), 1694 (w), 1628 and
1564 (amide); lmax (MeOH)/nm 207, 255 and 272 (ε 111,074, 22,425
and 23,532); lmax (Hþ) 208 and 272; lmax (OHꢀ) 210 and 274; dH
(500 MHz, C2HCl3þC2H3O2H, 223 K) 0.88 (9H, s, SiC(CH3)3), 3.4e3.8
(4H, m, H-7 and H-9), 4.40 (1H, d, JB,A 15.5, NCH2Ph), 4.49 (1H, m, H-
6), 4.97 (1H, d, JA,B 15.5, NCH2Ph), 7.07e7.54 (15H, ArH) and 8.13
(1H, s, NCHO). A minor by-product with very close resemblance to
the major product was clearly present with a signal at 7.99 ppm
(0.4H, s, NCHO), which was not due to a rotamer, since saturation
transfer was not be observed with the singlet at 8.13 ppm. Corre-
lation spectroscopy (COSY) was used to assign the signals from 3.4
to 4.99 ppm. This showed the coupling of the signals from the major
product. The mass spectrum was recorded again after 3 weeks in
deuteriochloroform and displayed no [MþH]þ ion at m/z 582 for
the diformylated by-product, suggesting loss the second formyl
group after storage in the acidic solvent.
4.19. (5S)-3-Benzyl-5-(O-methoxymethoxymethyl)-oxazolidin-
2-one (35)
Phosphorus pentoxide (5.0 g, 35.2 mmol) was added to a solu-
tion of (5S)-3-benzyl-5-hydroxymethyloxazolidin-2-one 34
(1.908 g, 9.2 mmol) and dimethoxymethane (25 mL, 282 mmol) in
chloroform (25 mL). The mixture was stirred at room temperature
for 1.5 h and decanted into ice-cool saturated aqueous sodium
carbonate (50 mL). The resulting mixture was extracted with ethyl
acetate (3ꢂ25 mL). The combined organic extracts were dried
(MgSO4) and the solvent was removed in vacuo. Flash chromatog-
raphy on silica gel using ethyl acetate/petroleum ether (7:3) as el-
uent
afforded
(5S)-3-benzyl-5-(O-methoxymethoxymethyl)-
oxazolidin-2-one 35 (1.819 g, 79%) as a colourless oil; ½a D23
þ17.2 (c
ꢃ
1, CHCl3); m/z (ammonia CI) found 252.1235, [C13H17NO4þH]þ re-
quires 252.1236; m/z [FAB, (3-NBA)] 252 ([MþH]þ); nmax (film)/
cmꢀ1 1746 (OCON); dH (300 MHz, C2HCl3) 3.26e3.36 (1H, m, H-4A),
3.31 (3H, s, OCH3), 3.35e3.51 (1H, m, H-4B), 3.60e3.71 (2H, m, H-6),
4.44 (2H, m, CH2Ph), 4.60e4.68 (1H, m, H-5), 4.62 (2H, s, OCH2O)
and 7.28e7.39 (5H, m, ArH); dC (75.48 MHz, C2HCl3) 45.8 (C-4), 48.2
(NCH2Ph), 55.4 (OCH3), 67.9 (C-6), 71.6 (C-5), 96.7 (OCH2O), 128.1,
128.8, and 135.7 (Ar) and 157.8 (C-2).
4.17. Methyl (5S)-3-benzyloxazolidin-2-one-5-carboxylate (33)
Butyl lithium (10.8 mL of a 1.6 M solution, 17.28 mmol) was
added to a solution of diisopropylamine (2.4 mL, 17.2 mmol) in
tetrahydrofuran (9 mL) at ꢀ78 ꢁC. The solution was allowed to
warm to 0 ꢁC and stirred for 15 min before being transferred via
cannula to
a
solution of methyl (5S)-oxazolidin-2-one-5-
carboxylate 21 (2.240 g, 15.45 mmol) in tetrahydrofuran (80 mL)
at ꢀ78 ꢁC. Hexamethylphosphoramide (10.8 mL) was added and
the solution was warmed to ꢀ30 ꢁC at which it was maintained for
30 min. The solution was cooled to ꢀ78 ꢁC and a solution of benzyl
bromide (2.4 mL, 20.18 mmol) in tetrahydrofuran (9 mL) was added.
The solution was stirred at ꢀ78 ꢁC for 2 h, allowed to warm to room
temperature and stirred for 2 h. The solvent was removed in vacuo
to yield an orange oil. Flash chromatography on silica gel using
ethyl acetate/petroleum ether (2:3) as eluent yielded methyl (5S)-
3-benzyloxazolidin-2-one-5-carboxylate 33 (1.844 g, 51%) as
4.20. (2S)-1-Benzylamino-3-(methoxymethoxy)-propan-2-ol
(36)
A
solution of (5S)-3-benzyl-5-(O-methoxymethoxymethyl)-
oxazolidin-2-one 35 (531 mg, 2.116 mmol) in methanol (10 mL) was
heated at reflux for 12 h with aqueous sodium hydroxide (2 M,
10 mL, 20 mmol). The methanol was removed in vacuo and the
residue was extracted with ethyl acetate (3ꢂ25 mL). The combined
organic extracts were dried (MgSO4) and the solvent was removed
in vacuo. Flash chromatography on silica gel using ethyl acetate as

7228
S.J. Baker et al. / Tetrahedron 70 (2014) 7221e7228
eluent
afforded
(2S)-1-benzylamino-3-(methoxymethyoxy)-
dichloromethane (2.5 mL) at 0 ꢁC. After 10 min TLC (1:9, ethanol/
propan-2-ol 36 as a colourless oil (427 mg, 90%); ½a D23
ꢃ
ꢀ1.5 (c 1,
ethyl acetate) indicated no starting material remaining and a single
higher running product. Methanol (20 mL) was added, followed by
10% palladium on charcoal (400 mg). The mixture was stirred under
an atmosphere of hydrogen for 1.5 h and the solvent was removed in
vacuo. Pyridine (0.8 mL) was added and the mixture was cooled in
an ice bath. Freshly prepared7 formic acetic anhydride (10 mL) was
added. When the effervescence ceased, the mixture was allowed to
warm to room temperature and stirred overnight. The mixture was
diluted with dichloromethane and filtered through CeliteÒ. The
solvent was removed in vacuo (high vacuum). Flash chromatogra-
phy on silica gel using ethanol/ethyl acetate (1:9) as eluent afforded
(6R)-2-(N,N-diformylamino)-8-benzyl-5-formyl-4-(3H)-oxo-6-
(methyloxymethyloxymethyl)-5,6,7,8-tetrahydropteridine 38 (36
CHCl3); m/z (ammonia CI) found 226.1442, [C12H19NO3þH]þ re-
quires 226.1443; m/z [FAB, 3-NBA] 226 ([MþH]þ); nmax (film)/cmꢀ1
3315 (OH and NH); dH (300 MHz, C2HCl3) 2.62e2.80 (2H, m, H-1),
2.82 (1H, br s, OH), 3.35 (3H, s, OCH3), 3.49e3.62 (2H, m, H-3), 3.80
(2H, m, NCH2Ph), 3.85e3.91 (1H, m, H-2), 4.62 (2H, s, OCH2O) and
7.23e7.25 (6H, m, ArH and NH); dC (75.48 MHz, C2HCl3) 51.8 (C-1),
54.2 (NCH2Ph), 55.7 (OCH3), 69.4 (C-2), 71.5 (C-3), 97.3 (OCH2O),
and 125.5, 128.6, 128.9 and 140.3 (Ar).
4.21. 2-Amino-6-((20S)-N-benzyl-30-(O-methoxymethylhy-
droxy)-20-hydroxypropylamino)-5-nitro-4-(3H)-pyrimidone
(37)
mg, 33%) as a white solid; mp 84e86 ꢁC; ½a D23
þ2.0 (c 0.91, CHCl3); m/
ꢃ
2-Amino-6-chloro-5-nitro-pyrimidin-4-(3H)-one 115 (190 mg,
1.0 mmol) was dissolved in methanol (22 mL) at 60 ꢁC. The pH was
adjusted to 9 by addition of triethylamine and a solution of (2S)-1-
benzylamino-3-(methoxymethoxy)-propan-2-ol 36 (223 mg,
0.991 mmol) in methanol (3 mL) was added. The pH was adjusted
to 9. The solution was heated at reflux for 7 h. The solvent
was removed in vacuo to afford a yellow foam. Flash chromatog-
raphy on silica gel using ethanol/ethyl acetate (1:9) as eluent yiel-
ded 2-amino-6-((20S)-N-benzyl-30-(O-methoxymethylhydroxy)-20-
hydroxypropylamino)-5-nitro-4-(3H)-pyrimidone 37 (302 mg,
z [FAB, (3-NBA)] 416 ([MþH]þ); m/z (EIþ) 415 [M]þ; nmax (KBr)/cmꢀ1
3424 (OH), 1678 (C]O) and 1638 (C]O); lmax (MeOH)/nm 274 and
250 (ε 2084 and 2460); lmax (Hþ) 294 and 252; lmax (OHe) 278 and
228; dH (500 MHz, C2HCl3) 3.30 (3H, s, OCH3), 3.71 (1H, dd, J9A,9B 9.8,
J9A,6 3.5, H-9A), 3.78 (1H, dd, J7A,7B 10.5, J7A,6 2.6, H-7A), 3.89 (1H, t,
J9B,9A¼J9B,6¼9.8, H-9B), 4.23 (1H, dd, J7B,7A 10.5, J7B,6 4.4, H-7B), 4.46
(2H, AB, JAB 16.2, NCH2Ph), 4.62 (2H, AB, JAB 6.5, OCH2O), 4.86 (1H,
dddd, J6,7A 4.4, J6,7B 2.6, J6,9A 9.8, J6,9B 3.5, H-6), 7.18e7.42 (5H, m,
ArH), 8.60 (1H, s, CHO), 8.70 (1H, s, CHO), 8.88 (1H, s, CHO) and 12.10
(1H br s, OH); dC (C2HCl3) 50.9 (C-7), 53.3 (NCH2Ph), 55.7 (C-6), 56.1
(OCH3), 66.0 (C-9), 97.1 (OCH2O), 125.4 (C-4a), 126.9, 129.0, 129.8
and 134.5 (Ar), 150.7 (C-8a) 152.5 (C-2), 153.8 (C-4), 163.7 (CHO),
164.0 (CHO) and 175.4 (CHO).
80%) as a yellow solid; mp 79e80 ꢁC; ½a D23
þ33.6 (c 1, CHCl3); m/z
ꢃ
(ammonia CI) found 380.1569, [C16H21N5O6þH]þ requires
380.1570; m/z [EIþ] 379 ([M]þ); nmax (KBr)/cmꢀ1 3320 (OH), 3219
(NH) and 1677 (C]O); lmax (MeOH)/nm 357, 285 and 253 (ε 4822,
6979 and 15,330); lmax (Hþ) 357, 285 and 253; lmax (OHꢀ) 368 and
250; dH (300 MHz, (C2H3)2CO) 3.10 (3H, s, OCH3), 3.22e3.42 (4H, m,
H-30 and H-10), 3.91e4.00 (1H, m, H-20), 4.10 (1H, br s, OH), 4.42
(2H, s, OCH2O), 4.54e4.88 (2H, m, NCH2Ph), 7.14e7.22 (5H, m, ArH)
and 10.64 (1H, br s, NH); dC (75.48 MHz, (C2H3)2CO, 324 K) 53.4 (C-
10), 54.8 (NCH2Ph), 55.4 (OCH3), 69.3 (C-20), 71.2 (C-30), 97.6
(OCH2O), 114.7 (C-5), 128.3, 128.8, 129.4 and 138.0 (Ar), 154.1 (C-6),
159.7 (C-2) and 162.4 (C-4).
Acknowledgements
We thank the BBSRC and EPSRC for financial assistance.
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Methanesulfonyl chloride (0.028 mL, 0.362 mmol) was added to
a solution of 2-amino-6-((20S)-N-benzyl-30-(O-methoxymethylhy-
droxy)-propylamino)-5-nitro-4-(3H)-pyrimidone 37 (100 mg,
0.264 mmol) and triethylamine (0.048 mL, 0.345 mmol) in