Modular Synthesis of Constrained Ethyl (cEt) Purine and Pyrimidine Nucleosides

Article abstract of DOI:10.1021/acs.joc.5b00607

(Chemical Equation Presented). A modular and scalable approach to pyrimidine- and purine-containing constrained ethyl (cEt) nucleosides is demonstrated. Minimizing stereochemical adjustments and protecting group manipulations, diacetone glucose is convert

Full text of DOI:10.1021/acs.joc.5b00607

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Note
Modular Synthesis of Constrained Ethyl
cEt) Purine and Pyrimidine Nucleosides
(
Helen Blade, Derek Bradley, Louis Diorazio, Timothy Evans, Barry R. Hayter, and Gareth P Howell
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b00607 • Publication Date (Web): 20 Apr 2015
Downloaded from http://pubs.acs.org on April 27, 2015
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Modular Synthesis of Constrained Ethyl (cEt) Purine and
Pyrimidine Nucleosides
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Helen Blade, Derek Bradley, Louis Diorazio, Timothy Evans, Barry R. Hayter, Gareth P. Howell*
*
Pharmaceutical Development, AstraZeneca, Charter Way, Macclesfield, SK10 2NA, United Kingdom
gareth.howell@astrazeneca.com
TOC Graphic
Abstract
A modular and scalable approach to pyrimidineꢀ and purineꢀcontaining constrained ethyl (cEt)
nucleosides is demonstrated. Minimising stereochemical adjustments and protecting group manipulations,
diacetone glucose is converted to two representative cEt nucleosides via a functionalised, common
intermediate. The retrosynthetic approach to this complex class of drug precursors offers clear benefits
over existing routes based on step count and throughput/efficiency.
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As newer technologies progress away from traditional DNAꢀbased platforms, there is a growing interest
1
in oligonucleotide therapeutics comprising novel nucleoside units. One such motif, introduced by ISIS
2
Pharmaceuticals, utilises a [2.2.1] tricyclic core as exemplified by the pyrimidine (1a) and purine (1b)
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cEt nucleosides shown in SCHEME 1. A number of cEtꢀbased oligonucleotides are currently undergoing
3,4
5
clinical investigation. The 4’ꢀsubstitution in the furanose core of 1 poses a significant challenge, and at
40+ steps, the synthetic burden of producing the (typically) four constituent cEt nucleosides for an
oligonucleotide drug represents one of the largest seen in the “small molecule” arena.
SCHEME 1. Synthesis of cEt nucleosides
The synthesis of an individual cEt nucleoside such as 1a can be achieved using short (10 steps from 5ꢀ
6
methyluridine), linear synthetic sequences as reported recently by Hanessian et al. For the largeꢀscale
manufacture of an oligonucleotide drug, however, multiple cEt nucleosides are required (as well as 1a
and 1b, there are various other reported cEt nucleosides comprising uracil, cytosine, methyl cytosine and
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2,7,8,9,10
adenine bases).
The summation of multiple 10ꢀstep linear sequences quickly becomes unwieldy for
commercial use and a modular approach would be expected to offer reduced overall stepꢀcount and
supply chain flexibility.
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The only demonstrated route to multiple cEt nucleosides (2→3→4→1; see SCHEME 1) was reported in
2
2010 and has since been modified allowing access to multiꢀkilogram quantities of cEt nucleosides 1. In
this current approach, diacetone allofuranose 3 (derived from diacetone glucose 2) is converted to
building block 4 in 13% yield over 12 steps and is subsequently functionalised to give cEt nucleosides 1.
Beginning in 2013, we have been investigating alternative synthetic routes to cEt nucleosides such as 1a
1
1
and 1b with a focus on fewer overall steps (23 currently to produce 1a-b from 2), and greater atom
economy (60% of the molecular mass of mesylate 4 is contained in the three protecting groups). A further
requirement for any new synthetic approach is the avoidance of constrained methyl (cMe) sideꢀproducts
of the type 6 shown in SCHEME 2. These cMe sideꢀproducts, being generated from residual aldehyde 5,
are closely related to the desired cEt structures 1 and are persistent and difficult to remove. Avoidance of
1
2
these sideꢀproducts would represent a significant improvement with respect to eventual drug purity.
SCHEME 2. Generation of cMe impurities
OTBDPS
OTBDPS
O 1'
1
2
. MeMgBr
. IBX
5
'
H
O
O
6'
O
3. LiBH4
4. MsCl MsO
4'
2'
O
NapO
NapO 3'
O
5
O
4
O
O
base
base
HO
HO
O
O 1a-b
HO
HO
6
We decided to investigate analogs of the mesylate 4 with alternative protecting groups at the 3’ꢀ and 5’ꢀ
6
positions. In keeping with results reported by Hanessian et al, we found that glycosidation of structures
such as 7 can be achieved in the absence of 3’ꢀprotecting groups as shown in SCHEME 3. Although this
is attractive from an efficiency perspective, the subsequent cEtꢀforming cyclisation (8→9) becomes
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markedly more complex with a competing oxetaneꢀforming pathway leading to structures such as 10. Due
to our concern that the eventual cEt oligonucleotide drug could be contaminated with closelyꢀrelated
1
2
oxetane glycosides (representing a safety and efficacy risk) we elected to continue with a dual, 3’,5’ꢀ
protecting group strategy using the same protecting group for both, such that they might be introduced
and removed jointly, improving efficiency and minimising step count.
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There are few protecting groups that are both (i) capable of withstanding the required
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glycosidation/cyclisation procedures and (ii) amenable to largeꢀscale use. Benzyl (Bn) groups were
selected with the expectation that hydrogenolysis could be carried out to complete the nucleoside
15
2
synthesis, avoiding the DDQ and HF deprotection procedures employed currently.
SCHEME 3. Synthesis using one protecting group
This required a synthetic route to mesylate 11 and our retrosynthetic approach is shown in SCHEME 4.
We were unable to identify a cheaper, more readily available starting material than diacetone glucose 2
and we envisaged that the pendant 6’(R)ꢀmesylate in 11 could be derived from epoxide 12 via terminal
hydride reduction and mesylation of the resulting secondary alcohol. Using this approach, the natural 6’ꢀ
1
6
stereochemistry of glucose is utilised without the need for stereochemical adjustments. Moreover,
retention of the 7’ꢀC atom from diacetone glucose 2 circumvents the homologation sequence used
2
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currently (SCHEME 2) thus avoiding cMe impurities of the type 6 highlighted earlier.
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SCHEME 4. Retrosynthetic analysis
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The epoxide 12 was expected to be obtainable from diol 13 which would require preꢀinstallation of the
’ꢀquaternary stereocentre using aldol chemistry with formaldehyde as an electrophile and ketone 14
4
(obtainable in one step from 2) as the enol nucleophile.
With the retrosynthetic plan in place, we began the forward synthesis with the oxidation of diacetone
1
7
glucose 2 to ketone 14 using a modified version of a literature procedure as shown in SCHEME 5. We
were expecting the aldol reaction of 14 to produce regioꢀ and stereochemical mixtures and only after
numerous screening experiments did we find that ketone 14 undergoes an unusually selective reaction
with aqueous formaldehyde in the presence of triethylamine using 2ꢀmethyltetrahydrofuran as solvent.
This process, a variant of which was reported during the preparation of this manuscript by Kuwahara et
18
al, gives the acetal 15 in 65% yield after crystallisation. Structural assignment of 15 was not
straightforward and single crystal xꢀray analysis was used to confirm the relative stereochemistry. We
were very interested to see that Kuwahara et al obtained mixtures of 15 (identified using 1D/2DꢀNMR)
18
and the expected aldol, suggesting that subtle reagent and solubility effects may be responsible for
determining the outcome of reaction. Acetal 15 did not require any specific deprotection and underwent
reduction with sodium borohydride to give diol 16 as a single stereoisomer in 87% yield. With safety and
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scalability in mind, double benzyl protection of diol 16 was carried out using a phase transfer catalytic
system to give acetonide 17 in 94% yield.
SCHEME 5. Synthesis of building block 9.
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a) NaOCl (aq), TEMPO, KBr, DCM, 30 °C, 1 h, 81%; b) CH O (aq), Et N, 2ꢀmethyltetrahydrofuran, rt,
2
3
+
ꢀ
1
6 h, 65% after recrystallisation; c) NaBH , MeOH, 0 °C, 87%; d) BnBr, [Bu N ][HSO ], NaOH (aq), 2ꢀ
4
4
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methyltetrahydrofuran, rt, 16 h, 94%; e) HCOOH, AcOH, H O, rt, 30 min, 94%; f) TsCl,
2
+
ꢀ
[
PhCH (Et) N ][Cl ], NaOH (aq), toluene, rt, 3 h, 93%; g) LiAlH , THF, ꢀ5°C – rt, 2.5 h, 92%; h) MsCl,
2 3 4
DMAP, MTBE, 0 °C, 1 h, 91%.
A number of approaches were attempted to achieve the selective removal of the primary acetonide in 17.
A procedure was developed using an aqueous mixture of formic and acetic acids which allowed diol 13 to
be isolated in good yield and purity after workꢀup. We found that conversion of 13 to epoxide 12 could be
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achieved using a modified 1ꢀpot tosylation/substitution procedure giving 12 in 93% yield. Full
characterisation (including single crystal xꢀray analysis) verified the structure and stereochemistry of 12.
Hydride reduction at the epoxide terminus and mesylation of the resulting secondary alcohol proceeded
smoothly giving 11 in 84% yield over 2 steps.
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With access to gram quantities of building block 11, synthesis of the desired cEt nucleosides 1a and 1b
was demonstrated using the two divergent sequences shown in SCHEME 6.
SCHEME 6. Synthesis of nucleosides 1a and 1b.
a) Ac O, H SO , EtOAc, rt, 24 h, quantitative; b) thymine, N,Oꢀbis(trimethylsilyl)acetamide, TMSꢀOTf,
2
2
4
toluene, 80 °C, 2 h, 82%; c) NaOH (s), MeOH, 40 °C, 1 h, 80%; d) H (60 psig), Pd(OH) /C, EtOH, 25
2
2
°C, 6 h, 92%; e) 2ꢀaminoꢀ6ꢀchloropurine, N,Oꢀbis(trimethylsilyl)acetamide, TMSOTf, toluene, 80 °C, 2 h,
t
6
4%; f) NaO Bu (s), 3ꢀhydroxypropionitrile, THF, 0 °C to rt, 16 h, 76%; g) isobutyric anhydride, Et N,
3
DMAP, toluene, 105 °C, 16 h, 96% h) HCOONH , Pd(OH) /C, isopropanol, 80 °C, 90 min, 82%.
4
2
Concomitant acetonide cleavage and acetylation gave 19 as a ~1:2 mixture of cisꢀ and transꢀ isomers.
Vorbrüggen glycosidation using typical conditions gave 20 and 22 with excellent selectivity at the 1’ꢀ
2
0
position (>95:5 anomeric ratio) by HPLC. Solvolysis of the 2’ꢀOAc groups in 20 and 22 and subsequent
2,6
cyclisation proved straightforward using typical conditions. Following amide formation for the purine
series (23→24), the final proof of the synthetic approach was demonstrated successfully. Conversion of
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1→1a was achieved using Pd(OH) supported on carbon (Evonik E101) under hydrogen at 60 psig.
2
The same conditions did not give complete conversion of 24→1b and a transfer hydrogenation using the
2
1
same catalyst and ammonium formate in isopropanol was used. From mesylate 11, pyrimidine 1a was
generated in 60% yield over four steps; the purine analog 1b (which comprises an extra amide forming
step) was generated in 38% yield over five steps.
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In summary, by comparison to the existing synthetic route to 1a and 1b, the approach above offers: i) a
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step count reduction from 23 to 17; ii) increased yield to both 1a (19% versus 8%) and 1b (12% versus
6
%); iii) greater atom efficiency with building block 11 (493 Da) having 29% lower molecular mass than
4
(691 Da) owing to the replacement of the protecting groups and iv) an improved purity profile with the
avoidance of the related cMe analogs 6. We are confident that this approach constitutes a general method
for the synthesis of pyrimidine and purine cEt nucleosides and will ultimately help provide new
oligonucleotide medicines to patients in the near future.
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Experimental section
General Procedure. All of the processes described were carried out under nitrogen atmospheres without
the requirement for ovenꢀ or flameꢀdried glassware. All solvents and reagents were used as supplied
without any further treatment. Column chromatography was carried using automated systems with preꢀ
packed silica columns. NMR spectra were recorded at 400 or 500 MHz and calibrated using residual
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undeuterated solvent as an internal reference: CHCl , δ 7.24 ppm; DMSO, δ 2.49 ppm; MeOH, δ 3.30
3
ppm and reported in parts per million relative to trimethylsilane at δ 0.00 ppm. NMR data is reported in
the following format: chemical shift (multiplicity, coupling constant (Hz), integration) using the
following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, spt = septet, m = multiplet, br =
broad, dd = doublet of doublets, dt = doublet of triplets, ddt = doublet of doublet of triplets. High
resolution mass spectra (HRMS) were recorded on a quadrupoleꢀTOF mass spectrometer using positive
electrospray ionization.
(
3aR,5R,6aS)-5-[(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-3a,6a-dihydrofuro[2,3-
d][1,3]dioxol-6-one (14)
To a solution of diacetone glucose 2 (322 g, 1.24 mol) in DCM (1.60 L) was added KBr (1.48 g, 124
mmol) and TEMPO (4.83 g, 30.9 mmol). The resulting mixture was heated to 30 °C with stirring and
NaOCl (~15% aq, 768 mL, 1.55 mol) was added over ~1 h maintaining a temperature of 25 – 35 °C. Note
that additions carried out at lower temperatures can lead to accumulation of reactive mixtures and the
potential for thermal runaway. The layers were separated and the organic layer was washed sequentially
with a solution of KI (12.9 g, 77.6 mmol) in HCl (0.50 M aq, 805 mL), Na S O (aq, sat, 805 mL) and
2
2
3
NaHCO (~5% w/w aq, 805 mL). The combined organics were dried over MgSO and concentrated under
3
4
reduced pressure. The residue was azeodried three times by evaporation from toluene (500 mL) to give
1
ketone 14 (261 g, 81%) as a brown liquid: H NMR (400MHz, CDCl ) δ 6.15 (d, J = 4.5 Hz, 1H),
3
4.42 ꢀ 4.33 (m, 3H), 4.07 ꢀ 4.01 (m, 2H), 1.47 (s, 3H), 1.45 ꢀ 1.43 (m, 3H), 1.35 (s, 6H) consistent
17
with literature values.
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(
[
3aS,3bR,7aS,8aR)-7a-[(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyltetrahydro-3bH-
1,3]dioxolo[4,5]furo[3,2-d][1,3]dioxin-3b-ol (15)
To a solution of ketone 14 (39.8 g, 154 mmol) in 2ꢀmethyltetrahydrofuran (800 mL) was added Et N (215
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mL, 1.54 mol) and HCHO (37% w/w aq, 398 mL, 14.7 mol). The mixture was stirred at rt for 16 h. The
mixture was adjusted to pH 4 using NH Cl (aq, sat). After addition of EtOAc (200 mL) and H O (200
4
2
mL) the layers were separated. The aqueous layer was extracted twice using EtOAc (200 mL). The
combined organics were dried over MgSO and concentrated under reduced pressure. The residue was
4
dissolved into EtOAc (130 mL) at 70 °C and hexane (400 mL) was added dropꢀwise over 30 min. The
resulting turbid solution was allowed to cool to rt and left to stir for 16 h. The solid was collected via
filtration and the filter cake was washed twice with hexane (400 mL). The solid was dried inꢀvacuo at 40
1
°C for 16 h to give acetal 15 (32.0 g, 65%) as a light yellow crystalline solid: mp 102.0 – 104.5 °C; H
NMR (500 MHz, CDCl ) δ 5.99 (d, J = 4.3 Hz, 1H), 4.95 (d, J = 6.2 Hz, 1H), 4.85 (dd, J = 0.8, 6.0 Hz,
3
1
H), 4.82 (t, J = 6.9 Hz, 1H), 4.31 (d, J = 4.3 Hz, 1H), 4.15 (d, J = 13.0 Hz, 1H), 4.07 (d, J = 6.7 Hz,
13
2
H), 3.78 (d, J = 13.2 Hz, 1H), 3.68 (s, 1H), 1.62 (s, 3H), 1.43 (s, 3H), 1.35 (s, 3H), 1.33 (s, 3H);
C
NMR (125 MHz, CDCl ) δ 113.1, 108.4, 103.9, 99.8, 86.7, 83.8, 82.2, 76.4, 65.8, 65.0, 26.1, 25.9, 25.8,
3
+
23.8; HRMS (ESIꢀTOF) calcd for C H NaO [M + Na] m/z = 341.1207, found 341.1205. A crystal of
14
22
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15 with approximate dimensions of 0.20×0.30×0.80 mm was mounted and automatically centered on a
benchtop crystallographic system. Intensity measurements were performed using monochromated (doubly
curved silicon crystal) MoK radiation (0.71073 Å) from a sealed microfocus tube. Generator settings
α
were 50 kV, 1 mA. Data collection temperature was ꢀ73 °C. APEX2 software was used for preliminary
determination of the unit cell. Determination of integrated intensities and unit cell refinement were
performed using SAINT. The integration of the data yielded a total of 6381 reflections to a maximum θ
angle of 24.39° (0.86 Å resolution). The constants for the triclinic unit cell are a = 6.098(4) Å, b =
3
7
.378(5) Å, c = 9.239(5) Å, α = 106.18(2)°, β = 100.896(19)°, γ = 96.42(2)°, V = 386.0(4) Å .
These are based upon the refinement of the XYZꢀcentroids of reflections above 20 σ(I). The calculated
minimum and maximum transmission coefficients (based on crystal size) are 0.9150 and 0.9780. The
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average residual for symmetry equivalent reflections is Rint = 7.66% and R = 10.28%. XPREP
σ
determined the space group to be P 1, with Z = 1 for the formula unit,C H O . The structure was solved
1
4
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with XS and subsequent structure refinements were performed with XL. The final anisotropic fullꢀmatrix
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leastꢀsquares refinement on F with 204 variables converged at R = 6.07% for the observed data and
o
1
wR = 16.68% for all data. The goodnessꢀofꢀfit was 0.971. The largest peak on the final difference
2
ꢀ
3
ꢀ
3
electron density synthesis was 0.23 e /Å and the deepest hole was ꢀ0.34 e /Å with an RMS deviation of
ꢀ
3
3
0
.06 e /Å . On the basis of the final model, the calculated density is 1.369 g/cm and F(000) = 170.
Absolute stereochemistry was assigned relative to the (1’R,2’R) stereocentres derived from the parent
structure diacetone glucose 2.
(3aR,5R,6S,6aR)-5-[(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-5-(hydroxymethyl)-2,2-dimethyl-6,6a-
dihydro-3aH-furo[2,3-d][1,3]dioxol-6-ol (16)
A suspension of acetal 15 (204 g, 640 mmol) in MeOH (2.90 L) was cooled to 0 °C. Maintaining a
temperature below 8 °C, NaBH (22.9g, 605 mmol) was added portionꢀwise over 65 min. The resulting
4
solution was stirred for 30 min at 0 °C, and the reaction was then quenched by careful addition of H O
2
(
20.0 mL). Further H O (2.70 L) was added and the resulting solution extracted four times with DCM
2
(2.50 L). The combined organics were dried over MgSO and concentrated under reduced pressure to give
4
1
diol 16 (171 g, 87%) as an offꢀwhite solid: H NMR (500 MHz, CDCl ) δ 5.92 (d, J = 4.1 Hz, 1H), 4.73
3
(
dd, J = 4.1, 6.4 Hz, 1H), 4.59 (dd, J = 6.5, 7.5 Hz, 1H), 4.33 (t, J = 6.5 Hz, 1H), 4.16 (dd, J = 7.5, 9.3
Hz, 1H), 3.91 (dd, J = 6.5, 9.3 Hz, 1H), 3.79 (d, J = 11.9 Hz, 1H), 3.61 (d, J = 11.5 Hz, 1H), 2.80 (d, J =
6.8 Hz, 1H), 2.21 ꢀ 2.05 (m, 1H), 1.75 ꢀ 1.66 (m, 1H), 1.63 (s, 3H), 1.46 (s, 3H), 1.41 (s, 3H), 1.35 (s,
1
3
3
H); C NMR (125 MHz, CDCl ) δ 114.2, 108.9, 105.6, 90.5, 80.4, 77.5, 71.6, 65.8, 62.7, 27.0, 26.9,
3
+
26.0, 24.1; HRMS (ESIꢀTOF) calcd for C H NaO [M + Na] m/z = 313.1258, found 313.1257.
1
3
22
7
(3aR,5S,6S,6aR)-6-Benzyloxy-5-(benzyloxymethyl)-5-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-
dimethyl-6,6a-dihydro-3aH-furo[2,3-d][1,3]dioxole (17)
Diol 16 (1.03 g, 3.55 mmol) and tetrabutylammonium hydrogensulfate (0.24 g, 0.71 mmol) were
dissolved in 2ꢀmethyltetrahydrofuran (10.3 mL). Following addition of NaOH (~50% w/w aq, 1.86 mL,
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5.2 mmol) BnBr (0.86 mL, 7.22 mmol) was added dropꢀwise over 30 min. The reaction mixture was
stirred rapidly for 16 h. After addition of H O (5.10 mL), the phases were separated. The aqueous phase
2
was extracted twice with MTBE (10.3 mL) and the combined organics were washed with NaCl (aq, sat,
0
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5.10 mL), dried over MgSO and concentrated under reduced pressure to give acetonide 17 (1.56g, 94%)
4
1
as a clear oil: H NMR (500 MHz, CDCl ) δ 7.37 ꢀ 7.26 (m, 10H), 5.85 (d, J = 3.8 Hz, 1H), 4.80 (dd, J =
3
6.5, 7.5 Hz, 1H), 4.77 (d, J = 11.5 Hz, 1H), 4.69 (dd, J = 3.9, 5.2 Hz, 1H), 4.55 (d, J = 11.8 Hz, 1H), 4.47
(
d, J = 11.9 Hz, 1H), 4.43 (d, J = 11.5 Hz, 1H), 4.27 (d, J = 5.3 Hz, 1H), 4.07 (dd, J = 7.6, 9.2 Hz, 1H),
3
.79 (dd, J = 6.4, 9.2 Hz, 1H), 3.69 (d, J = 10.5 Hz, 1H), 3.59 (d, J = 10.5 Hz, 1H), 1.62 (s, 3H), 1.41 (s,
13
3H), 1.39 (s, 3H), 1.31 (s, 3H); C NMR (125 MHz, CDCl ) δ 138.1, 137.5, 128.5, 128.4, 128.1, 128.0,
3
127.7, 127.6, 114.1, 108.3, 105.4, 88.8, 80.4, 78.5, 77.4, 73.9, 72.9, 70.1, 66.1, 27.2, 27.1, 26.0, 23.9;
+
HRMS (ESIꢀTOF) calcd for C H NaO [M + Na] m/z = 493.2197, found 493.2196.
27
34
7
3
-O-Benzyl-4-[(benzyloxy)methyl]-1,2-O-(1-methylethylidene)-α-D-gulofuranose (13)
Acetonide 17 (2.00 g, 4.25 mmol) was dissolved in a preꢀmade mixture of AcOH (11.6 mL, 203 mmol),
HCOOH (4.80 mL, 127 mmol) and H O (3.60 mL, 200 mmol) and the mixture was stirred at rt for 30
2
min. The mixture was poured on to iceꢀcold NaOH (~50% w/w aq, 26.9 mL, 350 mmol) and stirred at 0
°C for 10 min. After addition of DCM (100 mL) and H O (100 mL) the layers were separated. The
2
aqueous layer was extracted twice with DCM (100 mL). The combined organics were dried over MgSO4
and concentrated under reduced pressure. The residue was purified by column chromatography on silica
1
gel (30% ꢀ 50% EtOAc in hexane) to give diol 13 (1.72 g, 94%) as a colourless oil: H NMR (500 MHz,
CDCl ) δ 7.36 ꢀ 7.24 (m, 10H), 5.81 (d, J = 3.8 Hz, 1H), 4.81 (d, J = 11.8 Hz, 1H), 4.67 (dd, J = 3.9, 5.3
3
Hz, 1H), 4.50 (d, J = 11.6 Hz, 2H), 4.42 (d, J = 11.9 Hz, 1H), 4.34 (d, J = 5.4 Hz, 1H), 4.23 (ddd, J =
2.5, 3.9, 6.4 Hz, 1H), 3.78 (ddd, J = 3.9, 7.9, 11.3 Hz, 1H), 3.69 (ddd, J = 4.4, 6.2, 10.9 Hz, 1H), 3.48 (q,
13
J = 10.2 Hz, 2H), 3.22 (d, J = 2.3 Hz, 1H), 2.43 (dd, J = 4.5, 7.9 Hz, 1H), 1.66 (s, 3H), 1.37 (s, 3H);
C
NMR (125 MHz, CDCl ) δ 137.7, 137.1, 128.6, 128.5, 128.2, 127.9, 127.8, 127.7, 114.3, 104.8, 88.4,
3
+
79.1, 79.1, 73.7, 72.9, 71.5, 71.2, 62.7, 26.7, 26.5; HRMS (ESIꢀTOF) calcd for C H NaO [M + Na]
24 30 7
m/z = 453.1884, found 453.1880.
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,6-Anhydro-3-O-benzyl-4-[(benzyloxy)methyl]-1,2-O-(1-methylethylidene)-α-D-gulofuranose (12)
To a solution of diol 13 (5.99 g, 13.9 mmol) and benzyltriethylammonium chloride (317 mg, 1.39 mmol)
in toluene (120 mL) was added NaOH (~50% w/w aq, 47.9 mL, 908 mmol). A solution of TsCl (2.84 g,
14.7 mmol) in toluene (120 mL) was added dropꢀwise over a period of ~2 h. The mixture was stirred for a
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further 1 h before H O (120 mL) was added and the layers were separated. The aqueous layer was
2
extracted twice with toluene (120 mL). The combined organics were washed with H O (60.0 mL), NaCl
2
(
aq, sat, 60.0 mL), dried over MgSO and concentrated under reduced pressure to give epoxide 12 (5.36 g,
4
1
9
3%) as a colourless crystalline solid: mp 72.0 – 72.5 °C; H NMR (400 MHz, CDCl ) δ 7.34 ꢀ 7.22 (m,
3
10H), 5.76 (d, J = 3.7 Hz, 1H), 4.79 (d, J = 12.3 Hz, 1H), 4.63 (dd, J = 3.8, 5.0 Hz, 1H), 4.56 (d, J = 12.2
Hz, 1H), 4.49 (d, J = 12.0 Hz, 1H), 4.41 (d, J = 12.0 Hz, 1H), 4.31 (d, J = 5.0 Hz, 1H), 3.53 (dd, J = 2.9,
4.2 Hz, 1H), 3.38 (d, J = 10.5 Hz, 1H), 3.24 (d, J = 10.5 Hz, 1H), 2.78 ꢀ 2.73 (m, 2H), 1.67 (s, 3H), 1.36
13
(
s, 3H); C NMR (125 MHz, CDCl ) δ 137.9, 137.8, 128.4, 127.9, 127.8, 127.7, 127.6, 114.0, 104.5,
3
8
5.8, 79.5, 78.7, 73.7, 72.6, 69.8, 53.0, 44.6, 26.9, 26.5; HRMS (ESIꢀTOF) calcd for C H NaO [M +
24 28 6
+
Na] m/z = 435.1778, found 435.1781.
A crystal of 12 with approximate dimensions of 0.20×0.60×1.00 mm was mounted and automatically
centred on a benchꢀtop crystallographic system. Intensity measurements were performed using
monochromated (doubly curved silicon crystal) MoK radiation (0.71073 Å) from a sealed microfocus
α
tube. Generator settings were 50 kV, 1 mA. Data collection temperature was ꢀ73 °C. APEX2 software
was used for preliminary determination of the unit cell. Determination of integrated intensities and unit
cell refinement were performed using SAINT. The integration of the data yielded a total of 12079
reflections to a maximum θ angle of 25.07° (0.84 Å resolution). The constants for the monoclinic unit cell
3
are a = 10.6029(15) Å, b = 8.4121(11) Å, c = 13.0245(17) Å, β = 109.451(5)°, V = 1095.4(3) Å .They are
based upon the refinement of the XYZꢀcentroids of 3851 reflections above 20.0 I/σ(I) with 2.93° ≤ θ
≤
23.20°. Data were corrected for absorption effects with SADABS using the multiꢀscan technique. The
ratio of minimum to maximum apparent transmission is 64.6:100.The average residual for symmetry
equivalent reflections is R = 5.24% and R = 4.84%. XPREP determined the space group to be P 1 21 1,
int
σ
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with Z = 2 for the formula unit, C H O . The structure was solved with SHELXTL XT and subsequent
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structure refinements were performed with XShell. The final anisotropic fullꢀmatrix leastꢀsquares
2
refinement on F with 273 variables converged at R = 3.79% for the observed data and wR = 12.85%
o
1
2
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for all data. The goodnessꢀofꢀfit was 0.847. The largest peak on the final difference electron density
ꢀ
3
ꢀ
3
ꢀ
3
synthesis was 0.30 e /Å and the deepest hole wasꢀ0.26 e /Å with an RMS deviation of 0.09 e /Å . On the
3
basis of the final model, the calculated density is 1.251 g/cm and F(000) = 440. Absolute stereochemistry
was assigned relative to the (1’R,2’R) stereocentres derived from the parent structure diacetone glucose 2.
3-O-Benzyl-4-[(benzyloxy)methyl]-6-deoxy-1,2-O-(1-methylethylidene)-α-D-gulofuranose (18)
Epoxide 12 (3.56 g, 8.63 mmol) was dissolved in THF (66.8 mL, 820 mmol) and the solution was cooled
to ꢀ5 °C. Dropꢀwise addition of LiAlH solution (~1 M in THF, 6.88 mL, 6.88 mmol) was carried out over
4
15 min and the mixture was stirred at ꢀ5 °C for a further 10 min. The mixture was allowed to warm to rt
and was stirred for 2 h. The mixture was cooled to 0 °C and H O (0.26 mL) was added then the mixture
2
was stirred for a further 5 min. After the addition of NaOH (~15% w/w aq, 0.26 mL), H O (0.79 mL) was
2
added and the resulting solution was allowed to warm to rt and stirred for 2 h. After addition of MgSO4
(3.56 g, 29.6 mmol) the mixture was stirred for 30 min. The resulting suspension was filtered through a
pad of Celite, and the filter cake was washed with EtOAc (20.0 mL). The filtrate was concentrated under
1
reduced pressure to give alcohol 18 (3.17 g, 92%) as a colourless solid: H NMR (400 MHz, CDCl ) δ
3
7
.36 ꢀ 7.24 (m, 10H), 5.82 (d, J = 3.9 Hz, 1H), 4.80 (d, J = 11.9 Hz, 1H), 4.68 (dd, J = 4.0, 5.4 Hz, 1H),
.53 (d, J = 6.7 Hz, 1H), 4.50 (d, J = 6.6 Hz, 1H), 4.42 (d, J = 12.0 Hz, 1H), 4.37 ꢀ 4.30 (m, 1H), 3.54 (d,
4
J = 10.1 Hz, 1H), 3.45 (d, J = 10.0 Hz, 1H), 3.08 ꢀ 3.00 (m, 1H), 1.64 (s, 3H), 1.37 (s, 3H), 1.20 (d, J =
13
6.5 Hz, 3H); C NMR (125 MHz, CDCl ) δ 138.0, 137.6, 128.5, 128.4, 128.0, 127.8, 127.7, 127.6, 114.1,
3
104.5, 89.2, 79.5, 78.8, 73.7, 72.8, 70.6, 67.2, 26.8, 26.6, 17.1; HRMS (ESIꢀTOF) calcd for C H NaO
6
2
4
30
+
[
M + Na] m/z = 437.1935, found 437.1932.
3-O-Benzyl-4-[(benzyloxy)methyl]-6-deoxy-1,2-O-(1-methylethylidene)-5-O-(methylsulfonyl)-α-D-
gulofuranose (11)
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To a solution of alcohol 18 (1.14 g, 2.75 mmol) and DMAP (52.0 mg, 0.41 mmol) in MTBE (6.00 mL)
was added Et N (0.77 mL, 5.50 mmol) and the resulting solution was cooled to 0 °C. Methanesulfonyl
3
chloride (0.32 mL, 4.13 mmol) was added dropꢀwise over 5 min and the reaction was stirred at 0 °C for 1
10
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h. After addition of MTBE (1.25 mL) and H O (1.25 mL), the layers were separated. The aqueous layer
2
was extracted twice with MTBE (5.00 mL). The combined organics were washed twice with H O (5.00
2
mL), NaCl (aq, sat, 5 mL), dried over magnesium sulfate and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (20% ꢀ 30% EtOAc in hexane) to give
1
mesylate 11 (1.24 g, 91%) as a colourless oil: H NMR (400 MHz, CDCl ) δ 7.39 ꢀ 7.22 (m, 10H), 5.87
3
(
d, J = 4.3 Hz, 1H), 5.25 (q, J = 6.6 Hz, 1H), 4.84 ꢀ 4.74 (m, 2H), 4.55 (d, J = 11.9 Hz, 1H), 4.45 ꢀ 4.42
(m, 2H), 4.20 (d, J = 5.5 Hz, 1H), 3.68 (d, J = 10.1 Hz, 1H), 3.61 (d, J = 10.1 Hz, 1H), 3.14 (s, 3H), 1.63
1
3
(s, 3H), 1.46 (d, J = 6.6 Hz, 3H), 1.38 (s, 3H); C NMR (125 MHz, CDCl ) δ 137.6, 137.1, 128.5, 128.5,
3
128.1, 128.0, 127.7, 114.3, 105.4, 88.3, 82.6, 80.6, 78.3, 73.9, 73.1, 69.7, 38.7, 27.0, 26.5, 18.8; HRMS
+
(
ESIꢀTOF) calcd for C H NaO S [M + Na] m/z = 515.1710, found 515.1705.
25
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1,2-Di-O-acetyl-3-O-benzyl-4-[(benzyloxy)methyl]-6-deoxy-5-O-(methylsulfonyl)-D-gulofuranose
(
19)
To a solution of mesylate 11 (1.00 g, 2.03 mmol) in EtOAc (4.00 mL) was added Ac O (576 µL, 6.09
2
mmol) and the solution was cooled to 0 °C. After addition of H SO (~98%, 22.2 µL, 0.406 mmol) the
2
4
reaction mixture was allowed to warm to rt and stirred for 24 h. After addition of NaHCO (aq, sat, 2.00
3
mL) the mixture was stirred at rt for 10 min. The layers were separated and the aqueous layer was
extracted three times with EtOAc (5.00 mL). The combined organics were dried over MgSO and was
4
concentrated under reduced pressure to give acetate 19 (1.09 g, quantitative) as a colourless oil.
Compound isolated as a 2:1 mixture of 1’ꢀepimers. A sample was purified by column chromatography on
silica gel (10% ꢀ 50% EtOAc in hexane) to give samples of both isomers which were assigned based on
1
coupling constants, transꢀ19 H NMR (500 MHz, CDCl ) δ 7.37 ꢀ 7.23 (m, 10H), 6.15 (d, J = 1.4 Hz,
3
1H), 5.46 ꢀ 5.38 (m, 1H), 5.17 (q, J = 6.6 Hz) 1.4H), 4.60 (d, J = 11.2 Hz, 1H), 4.55 ꢀ 4.45 (m, 4H), 3.68
13
(
d, J = 10.2 Hz, 1H), 3.53 (d, J = 10.2 Hz, 1H), 3.02 (s, 3H), 2.14 (s, 3H), 1.45 (d, J = 6.6 Hz, 3H); C
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NMR (125 MHz, CDCl ) δ 169.8, 169.3, 137.6, 136.8, 128.6, 128.5, 128.3, 128.1, 127.9, 127.7, 97.5,
3
1
8
8.8, 81.0, 78.6, 74.6, 74.2, 73.6, 69.4, 38.5, 20.9, 20.8, 18.0; cisꢀ19 H NMR (500 MHz, CDCl ) δ 7.38 ꢀ
3
7.27 (m, 10H), 6.44 (d, J = 4.8 Hz, 1H), 5.46 ꢀ 5.38 (m, 1H), 5.25 ꢀ 5.15 (m, 1H), 4.73 (d, J = 11.5 Hz,
0
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1H), 4.61 (m, 1H), 4.55 ꢀ 4.45 (m, 3H), 4.30 (d, J = 5.3 Hz, 1H), 3.72 (d, J = 10.3 Hz, 1H), 3.57 (d, J =
1
3
10.3 Hz, 0.4H), 3.03 (s, 1.2H), 3.02 (s, 3H), 2.14 (s, 3H), 1.99 (s, 3H), 1.34 (d, J = 6.6 Hz, 3H); C NMR
(
125 MHz, CDCl ) δ 170.5, 169.9, 137.6, 137.3, 128.6, 128.4, 128.0, 127.8, 127.7, 127.1, 94.2, 89.5,
3
8
1.8, 78.1, 74.5, 73.9, 73.2, 69.3, 38.4, 21.2, 20.5, 18.4; HRMS (ESIꢀTOF) calcd for C H NaO S [M +
26 32 10
+
Na] m/z = 559.1608, found 559.1600.
-{2-O-Acetyl-3-O-benzyl-4-[(benzyloxy)methyl]-6-deoxy-5-O-(methylsulfonyl)-β-D-gulofuranosyl}-
1
5-methylpyrimidine-2,4(1H,3H)-dione (20)
To a solution of acetate 19 (1.00 g, 1.86 mmol) in MeCN (5.00 mL) were added thymine (285 mg, 2.24
mmol) and N,Oꢀbis(trimethylsilyl)acetamide (1.38 mL, 5.59 mmol) and the resulting mixture was heated
at 40 °C for 1 h. The mixture was cooled to rt and TMSOTf (452 µL, 2.42 mmol) was added before the
mixture was heated at 80 °C for 1 h. The reaction mixture was cooled to 0 °C and the pH was adjusted to
pH 6ꢀ7 using NaOH (15% w/w aq). After addition of EtOAc (10.0 mL) and H O (10.0 mL) the layers
2
were separated. The aqueous layer was extracted twice with EtOAc (10.0 mL). The combined organics
were washed with NaCl (aq, sat, 10.0 mL), dried over MgSO and concentrated under reduced pressure to
4
1
give pyrimidine 20 (0.92 g, 82%) as a colourless foam: H NMR (500MHz, CDCl ) δ 7.44 ꢀ 7.28 (m,
3
1
2H), 6.23 (d, J = 6.6 Hz, 1H), 5.64 (t, J = 6.1 Hz, 1H), 5.07 (q, J = 6.4 Hz, 1H), 4.68 (d, J = 10.9 Hz,
H), 4.60 (s, 2H), 4.50 (d, J = 10.9 Hz, 1H), 4.46 (d, J = 5.7 Hz, 1H), 3.79 (d, J = 9.9 Hz, 1H), 3.65 (d, J
1
1
3
= 9.9 Hz, 1H), 2.94 (s, 3H), 2.09 (s, 3H), 1.57 (s, 3H), 1.38 (d, J = 6.5 Hz, 3H); C NMR (125 MHz,
CDCl ) δ 170.4, 136.8, 136.6, 136.1, 128.8, 128.7, 128.5, 128.4, 128.2, 127.8, 124.1, 111.7, 87.8, 87.0,
3
8
1
5
0.1, 79.1, 75.1, 74.5, 74.1, 70.2, 38.22, 20.8, 17.4, 12.1.
-[(1R,3S,4S,6R,7S)-7-Benzyloxy-4-(benzyloxymethyl)-3-methyl-2,5-dioxabicyclo[2.2.1]heptan-6-yl]-
-methyl-pyrimidine-2,4-dione (21)
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To a solution of pyrimidine 20 (1.10 g, 1.83 mmol) in MeOH (4.40 mL) was added NaOH (220 mg, 5.50
mmol) and the suspension was heated at 40 °C for 1 h. The volatiles were removed under reduced
pressure and the residue was taken up in EtOAc (10 ml) and H O (10.0 mL). The layers were separated
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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and the aqueous layer extracted twice with EtOAc (10.0 mL). The combined organics were washed with
NaCl (aq, sat, 10.0 mL), dried over MgSO and concentrated under reduced pressure to give pyrimidine
4
1
2
1
6
1 (678 mg, 80%) as a light yellow oil: H NMR (500 MHz, CDCl ) δ 8.29 (s, 1H), 7.45 (d, J = 1.0 Hz,
3
H), 7.41 ꢀ 7.28 (m, 10H), 5.61 (s, 1H), 4.64 (m, 3H), 4.56 (s, 1H), 4.50 (d, J = 11.6 Hz, 1H), 4.13 (q, J =
1
3
.7 Hz, 1H), 3.90 ꢀ 3.87 (m, 3H), 1.65 (d, J = 0.7 Hz, 3H), 1.32 (d, J = 6.7 Hz, 3H); C NMR (125 MHz,
CDCl ) δ 163.5, 150.0, 137.5, 136.8, 134.9, 128.6, 128.5, 128.1, 128.1, 127.9, 127.7, 110.1, 88.0, 87.0,
3
+
81.2, 73.9, 72.4, 64.9, 16.4, 12.4; HRMS (ESIꢀTOF) calcd for C H N O [M + H] m/z = 465.2020,
2
6
29
2
6
found 465.2022.
-[(1R,3S,4R,6R,7S)-7-Hydroxy-4-(hydroxymethyl)-3-methyl-2,5-dioxabicyclo[2.2.1]heptan-6-yl]-5-
1
methyl-pyrimidine-2,4-dione (1a)
To a solution of pyrimidine 21 (100 mg, 215 µmol) in EtOH (5.00 mL) was added Pd(OH) on carbon
2
(
Evonik type E101; 20% w/w loading, 27.5 mg) and the mixture was hydrogenated at 25 °C and 60 psig
for 6 h. The reaction mixture was filtered through a plug of Celite, washing through with EtOH (10.0
mL) and the filtrate was concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (0 ꢀ 10% MeOH in DCM) to give nucleoside 1a (56.0 mg, 92%) as a
1
colourless solid. All data in full agreement with authentic pharmaceutical samples: H NMR (400MHz,
MeOHꢀd ) δ 7.70 (s, 1H), 5.50 (s, 1H), 4.34 (s, 1H), 4.11 ꢀ 4.01 (m, 2H), 3.96 (s, 2H), 1.88 (s,
4
13
3
H), 1.32 (d, J = 6.7 Hz, 3H); C NMR (125 MHz, DMSOꢀd ) δ 163.9, 150.0, 134.9, 108.3. 89.0.
6
+
8
6.0. 80.3. 79.3. 69.6, 56.1, 16.3, 12.3; HRMS (ESIꢀTOF) calcd for C H N O [M + H] m/z =
1
2
17
2
6
285.1081, found 285.1087.
[
(2R,3R,4S,5R)-2-(2-Amino-6-chloro-purin-9-yl)-4-benzyloxy-5-(benzyloxymethyl)-5-[(1R)-1-
methylsulfonyloxyethyl]tetrahydrofuran-3-yl] acetate (22)
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To a suspension of acetate 19 (1.08 g, 2.01 mmol) in toluene (8.64 mL) were added 4ꢀaminoꢀ6ꢀ
chloropurine (375 mg, 2.21 mmol) and N,Oꢀbis(trimethylsilyl)acetamide (1.48 mL, 6.04 mmol) and the
mixture was heated at 80 °C for 1 h. The mixture was cooled to rt and TMSOTf (729 µL, 4.03 mmol) was
added dropꢀwise over 5 min and the mixture was heated to 80 °C for 70 min. The mixture was cooled to 0
°C and the pH was adjusted to pH 6ꢀ7 using NaOH (~15% w/w aq). After the addition of EtOAc (10.0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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9
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9
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mL) and H O (10.0 mL) the layers were separated. The aqueous layer was extracted twice with EtOAc
2
(
10.0 mL). The combined organics were washed with NaCl (aq, sat, 10.0 mL), dried over MgSO and
4
1
concentrated under reduced pressure to give purine 22 (0.83 g, 64%) as a colourless foam: H NMR (500
MHz, CDCl ) δ 7.92 (s, 1H), 7.43 ꢀ 7.29 (m, 10H), 6.22 (d, J = 6.3 Hz, 1H), 6.02 (t, J = 6.0 Hz, 1H), 5.22
3
ꢀ 5.19 (m, 3H), 4.66 (d, J = 10.9 Hz, 1H), 4.62 (d, J = 5.7 Hz, 1H), 4.59 ꢀ 4.53 (m, 3H), 3.71 (d, J = 10.0
1
3
Hz, 1H), 3.64 (d, J = 10.0 Hz, 1H), 2.90 (s, 3H), 2.03 (s, 3H), 1.41 (d, J = 6.6 Hz, 3H); C NMR (125
MHz, CDCl ) δ 171.2, 170.0, 159.2, 153.6, 151.5, 141.0, 136.9, 136.6, 128.8, 128.7, 128.5, 128.3, 128.2,
3
127.9, 125.4, 88.2, 85.4, 80.3, 79.1, 75.0, 74.8, 73.9, 69.7, 60.4, 38.5, 21.1, 20.7, 17.4; HRMS (ESIꢀTOF)
+
calcd for C H ClN O S [M + H] m/z = 646.1733, found 646.1730.
2
9
33
5
8
2-Amino-9-[(1R,3S,4S,6R,7S)-7-benzyloxy-4-(benzyloxymethyl)-3-methyl-2,5-
dioxabicyclo[2.2.1]heptan-6-yl]-1H-purin-6-one (23)
t
A solution of NaO Bu (700 mg, 7.26 mmol) in anhydrous THF (5.00 mL) was placed under an inert
atmosphere and cooled to 0 °C. After stirring for 10 min, 3ꢀhydroxypropionitrile (496 µL, 7.26 mmol)
was added dropꢀwise and the resulting mixture was stirred at 0 °C for 30 min. A solution of purine 22
(
816 mg, 1.26 mmol) in anhydrous THF (5.00 mL) was added dropꢀwise and the resulting mixture was
stirred at 0 °C for 1 h before warming to rt and stirring for 16 h. The reaction mixture was cooled to 0 °C
and H O (2.00 mL) was added dropꢀwise. After addition of EtOAc (10.0 mL), the layers were separated.
2
The organic layer was washed with NH Cl (aq, sat, 5.00 mL), dried over MgSO and concentrated under
4
4
reduced pressure. The residue was purified by column chromatography on silica gel (0% ꢀ 10% MeOH in
1
DCM) to give aniline 23 (472 mg, 76%) as a yellow solid: H NMR (500 MHz, DMSOꢀd ) δ 10.76 ꢀ
6
10.54 (m, 1H), 7.71 (s, 1H), 7.37 ꢀ 7.26 (m, 10H), 6.58 (br s, 2H), 5.73 (s, 1H), 4.72 (s, 1H), 4.67 ꢀ 4.57
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(
(
m, 4H), 4.28 (s, 1H), 4.13 (q, J = 6.6 Hz, 1H), 3.93 (d, J = 11.6 Hz, 1H), 3.88 (d, J = 11.5 Hz, 1H), 1.26
13
d, J = 6.6 Hz, 3H); C NMR (126 MHz, DMSOꢀd ) δ 156.7, 153.9, 150.5, 138.0, 137.8, 134.1, 128.3,
6
1
28.3, 127.6, 127.6, 127.6, 127.4, 116.7, 87.0, 84.4, 80.5, 78.7, 77.0, 72.8, 71.4, 65.6, 16.3; HRMS (ESIꢀ
+
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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59
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TOF) calcd for C H N O [M + H] m/z = 490.2085, found 490.2090.
26
28
5
5
N-[9-[(1R,3S,4S,6R,7S)-7-Benzyloxy-4-(benzyloxymethyl)-3-methyl-2,5-dioxabicyclo[2.2.1]heptan-6-
yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide (24)
To a solution of purine 23 (287 mg, 0.59 mmol) in toluene (2.00 mL) were added triethylamine (245 µL,
1.76 mmol) and DMAP (36.0 mg, 0.29 µmol). Isobutyric anhydride (291 µL, 1.76 mmol) was added and
the reaction mixture was heated at 105 °C for 16 h. The mixture was cooled to 40 °C, MeOH (175 µL)
was added and the resulting mixture was stirred at 40 °C for 30 min. The solution was cooled to rt, NH Cl
4
(aq, sat, 5.00 mL) was added and the mixture was stirred for 20 min. The layers were separated and the
aqueous layer was extracted twice with toluene (5.00 mL), dried over MgSO and concentrated under
4
1
reduced pressure to give amide 24 (316 mg, 96%) as a paleꢀbrown solid: H NMR (500 MHz, CDCl ) δ
3
12.01 (s, 1H), 8.55 (br s, 1H), 7.76 (s, 1H), 7.37 ꢀ 7.20 (m, 10H), 5.76 (s, 1H), 4.62 (s, 2H), 4.55 – 4.44
(
m, 3H), 4.21 (d, J = 6.7 Hz, 1H), 4.09 (s, 1H), 3.89 ꢀ 3.80 (m, 2H), 2.65 (quin, J = 6.9 Hz, 1H), 1.33 (d,
1
3
J = 6.7 Hz, 3H), 1.27 (dd, J = 4.3, 6.9 Hz, 7H); C NMR (126 MHz, CDCl ) δ 178.6, 155.5, 147.5,
3
147.0, 137.4, 136.8, 136.0, 128.6, 128.5, 128.1, 128.0, 127.7, 127.7, 121.8, 87.8, 85.9, 81.6, 78.3, 77.6,
+
7
3.9, 72.7, 65.1, 36.6, 19.0, 16.4; HRMS (ESIꢀTOF) calcd for C H N O [M + H] m/z = 560.2504,
3
0
34
5
6
found 560.2497.
N-[9-[(1R,3S,4R,6R,7S)-7-Hydroxy-4-(hydroxymethyl)-3-methyl-2,5-dioxabicyclo[2.2.1]heptan-6-
yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide (1b)
i
To a solution of amide 24 (38.6 mg, 0.07 mmol) in PrOH (4.00 mL) was added ammonium formate (250
mg, 3.97 mmol) and Pd(OH) on carbon (Evonik type E101; 20% w/w loading, 25 mg). The mixture was
2
sealed in a pressure tube and heated to 80 °C with stirring for 90 min. The mixture was cooled and the
i
catalyst removed via filtration through celite which was rinsed with PrOH (2.00 mL). The resulting
solution was concentrated inꢀvacuo and purified by column chromatography on silica gel (5% ꢀ 10%
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MeOH in DCM) to give nucleoside 1b (21.3, 82%) as a white solid. All data in full agreement with
1
authentic pharmaceutical samples: H NMR (400 MHz, MeOHꢀd ) δ 8.07 (s, 1H), 5.84 (s, 1H), 4.54 (s,
4
1H), 4.27 (s, 1H), 4.16 (q, J = 6.8 Hz, 1H), 4.00 (s, 2H), 2.71 (spt, J = 6.9 Hz, 1H), 1.37 (d, J = 6.8 Hz,
13
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
3H), 1.22 (d, J = 6.9 Hz, 6H); C NMR (101 MHz, MeOHꢀd ) δ 180.4, 156.0, 148.5, 148.0, 137.0, 120.2,
4
+
89.0, 86.0, 81.2, 80.1, 70.8, 56.8, 35.6, 17.9, 15.3; HRMS (ESIꢀTOF) calcd for C H N O [M + H] m/z
16
22
5
6
=
380.1565, found 380.1561.
1
13
Supporting Information. NMR spectra ( H and C) are provided for all synthesised compounds. HPLC
traces are provided for crude reaction mixtures of compounds 19, 20 and 22 to illustrate selectivity.
ORTEP diagrams and .cif files are provided for compounds 12 and 15. This material is available free of
charge via the Internet at http://pubs.acs.org.
Acknowledgements
The authors would like to thank Andy McPherson, Michael Migawa and Lijan Chen for useful
discussions along with Anthony Bristow and Helen Wheatcroft for analytical assistance.
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References and footnotes
1
2
Tamm, I.; Dörken, B.; Hartmann, G. Lancet 2001, 358, 489 – 497.
10
11
12
13
14
15
16
17
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19
20
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23
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59
60
Seth, P. P.; Vasques, G.; Allerson, C. A.; Berdeja, A.; Gaus, H.; Kinberger, G. A.; Prakash, T. P.;
Migawa, M. T.; Bhat, B.; Swayze, E. E. J. Org. Chem. 2010, 75, 1569 – 1581.
3
Gibson, N. W. J. R. Coll. Physicians Edinburgh 2014, 44, 196.
4
Hong, D.; Kim, Y.; Younes, A.; Nemunaitis, J.; Fowler, N.; Hsu, J.; Zhou, T.; Fayad, L.; Zhang, N.;
PihaꢀPaul, S.; Woessner, R.; Nadella, M. V.; Lawson, D.; Reimer, C.; Jo, M.; Schmidt, J.; Xiao, X.;
Greenlee, S.; Hung, G.; Yamashita, M.; Blakey, D. C.; Monia, B. P.; Macleod, A. R.; Kurzrock, R.
Cancer Res. 2014, 74, LBꢀ227.
5
Betson, M.; Allanson, N.; Wainwright, P. Org. Biomol. Chem. 2014, 12, 9291 – 9306.
6
Salinas, J. C.; Migawa, M. T.; Merner, B. L. Hanessian, S. J. Org. Chem. 2014, 79, 11651 – 11660.
7
Seth, P. P.; Yu, J.; Allerson, C. R.; Berdeja, A.; Swayze, E. E. Bioorg. Med. Chem. Lett. 2011, 21, 1122
–
1125.
8
Seth, P. P.; Siwkowski, A.; Allerson, C. R.; Vasquez, G.; Lee, S.; Prakash, T. P.; Kinberger, G.;
Migawa, M. T.; Gaus, H.; Bhat, B.; Swayze, E. E. Nucleic Acids Symp. Ser. 2008, 52, 553 – 554.
9
Vasquez, G. Patent WO 2009/143369 A2, 2009.
10
Swayze, E. E.; Seth, P. P. Patent WO 2007/090071 A2, 2007.
11
12
The 12 steps required to synthesise the common intermediate 4 are only counted once.
Defects in the modular sugar core undergo net amplification when converted into oligonucleotide
chains. For example, four nucleosides (A, G, C, U) derived from a common sugar (S) building block
containing x% of an anomalous sugar impurity (S*) will each contain x% of the related nucleoside
impurity (A*, G*, C*, U*). When multiple (n) nucleosides are coupled into an oligonucleotide sequence,
there will be a family of oligonucleotide impurities containing one (unspecified) anomalous nucleoside
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(
AꢀGꢀC…)* present at a level of nx%. The exact identity, as well as the toxicology and efficacy, of
specific impurities in this family require careful consideration.
13
For a survey of methods for glycosidation, see: Merino, P. Chemical Synthesis of Nucleoside
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
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9
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8
9
0
Analogues; Wiley: 2013.
1
4
The SELECT criteria, representing Safety, Environmental, Legal, Economy, Control and Throughput
are frequently used as criteria for successful scaleꢀup, see: Butters, M.; Catterick, D.; Craig, A.; Curzons,
A.; Dale, D.; Gillmore, A.; Green, S. P.; Marziano, I.; Sherlock, J.ꢀP.; White, W. Chem. Rev. 2006, 106,
3
002 – 3027.
15
Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.;
Wengel, J. Tetrahedron 1998, 54, 3607 – 3630.
16
The 6’ꢀepimer of epoxide 10, generated in 13 steps from 2, has been used previously for the synthesis
of nucleosides but is stereochemicallyꢀunsuitable for application to cEt structures, see:Enderlin, G.;
Nielsen, P. J. Org. Chem. 2008, 73, 6891 – 6894.
1
1
1
2
7
8
9
0
Turks, M.; Rodins, V. Rolava, E.; Ostrovskis, P.; Belyakov, S. Carbohydr. Res. 2013, 375, 5 – 15.
Fukuyama, K.; Ohrui, H.; Kuwahara, S. Org. Lett. 2015, 17, 828 – 831.
Szeja, W. Carbohydr. Res. 1986, 158, 245 – 248.
Vorbruggen, H.; RuhꢀPohlenz, C. Org. React. (Hoboken, NJ, U. S.) 2000, 55.
Cipolla, L.; Airoldi, C.; Bini, D.; Gregori, M.; Marcelo, F.; JiménezꢀBarbero, J.; Nicotra, F. Eur. J.
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Org. Chem. 2011, 128 – 136.
22
In keeping with the stepꢀcounting approach used previously (SCHEME 1 and ref [11], the 8 steps to
common intermediate 11 are only counted once.
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