Stereoselective preparation of (RP)-8-hetaryladenosine-3′, 5′-cyclic phosphorothioic acids

Article abstract of DOI:10.1039/b702403g

Cyclic adenosine monophosphate (cAMP) has been converted into its 8-bromo derivative and 2′O-TBDMS protected before activation of the phosphoric acid moiety with a reagent generated in situ from oxalyl chloride and DMF. Further reactions with primary amines furnished corresponding phosphoramidates with high stereoselectivity at the phosphorus atom. Cross-coupling reactions with the 8-bromopurine yielded 8-hetaryl derivatives. X-Ray analyses showed the amidates to possess the (S_P)-configuration. Carbon disulfide effected thiylation under strongly basic conditions stereospecifically provided the (R_P)-phosphorothioic acids. The Royal Society of Chemistry.

Full text of DOI:10.1039/b702403g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Stereoselective preparation of (RP)-8-hetaryladenosine-3ꢀ,5ꢀ-cyclic
phosphorothioic acids†
Mioara Andrei,a Vidar Bjørnstad,a,b Geir Langli,a Christian Rømming,c Jo Klaveness,b Kjetil Taske´na,d and
Kjell Undheim*a,c
Received 15th February 2007, Accepted 2nd May 2007
First published as an Advance Article on the web 29th May 2007
DOI: 10.1039/b702403g
Cyclic adenosine monophosphate (cAMP) has been converted into its 8-bromo derivative and
2ꢀO-TBDMS protected before activation of the phosphoric acid moiety with a reagent generated in situ
from oxalyl chloride and DMF. Further reactions with primary amines furnished corresponding
phosphoramidates with high stereoselectivity at the phosphorus atom. Cross-coupling reactions with
the 8-bromopurine yielded 8-hetaryl derivatives. X-Ray analyses showed the amidates to possess the
(SP)-configuration. Carbon disulfide effected thiylation under strongly basic conditions
stereospecifically provided the (RP)-phosphorothioic acids.
Introduction
The naturally occurring purine cyclic monophosphates, cyclic
adenosine monophosphate (cAMP) and cyclic guanosine
monophosphate (cGMP), are important secondary messengers,
regulating a wide range of cell functions in response to various
hormones.1,2 In one group of cAMP analogues, one of the oxygen
atoms pendant from the phosphorus atom has been replaced
by a sulfur atom. The new stereogenic center created at the
phosphorus atom provides an (RP)- and an (SP)-adenosine-3ꢀ,5ꢀ-
cyclic monophosphorothioic acid (cAMPS) diastereomer, which
differ in the pharmacological effect they cause at the level of the
Fig. 1 Target molecules.
effector, protein kinase A, where (RP)-cAMPS acts as a competitive
antagonist and (SP)-cAMPS acts as an agonist.3,4 Substitution
in cAMPS at the purine 8-position may affect bioactivity as
Target molecules
demonstrated for some 8-thia, -oxa and -aza derivatives.5,6 cAMPS
derivatives with a carbon substituent in the purine 8-position are
of interest as immune stimulating agents.7 Considerable efforts
have been devoted to studies of 8-halogeno derivatives,5,8–10 and
the 8-bromo analogue has been proposed as an immune stimulant
in HIV treatment.10,11
cAMPS derivatives carrying a carbon substituent in the 8-
position were not available when the present work was initiated.
We report a stereoselective and convenient preparative synthesis of
members of the (RP)-cAMPS family of cAMP analogues carrying
a carbon substituent in the 8-position. The methodology involves
stereoselective thiation at the phosphorus atom and introduction
of a carbon substituent into the purine 8-position in cAMPS
derivatives. The general structure for the target molecules is shown
in Fig. 1, structure A.
Reports are available on the preparation of the parent cAMPS
compound (structure A, R = H) and a few 8-hetero-substituted
derivatives. In most cases, adenosine has served as a substrate
for thiophosphorylation at the 5ꢀ-OH group by thiophosphoryl
chloride and a subsequent alkali hydroxide treatment to effect
cyclisation. The steric induction by this approach was poor,
however, and the diastereoisomers were separated by extensive
chromatographic operations.12 Other cyclothiophosphorylation
reactions reported also show a low degree of stereoselectivity and
require chromatographic separation steps for the isolation of the
diastereomers.13,14 The (RP)-diastereomer of cAMPS, however, has
been prepared enzymatically from the corresponding nucleoside
5ꢀO-(1-thiotriphosphate).15 A more promising approach appeared
to be the use of cAMP as a substrate. The latter was converted into
a phosphoryl chloride in an Apple type reaction using Ph3P–CCl4,
and the product reacted with amines to form phosphoramidates.
The reaction sequence proceeded with low diastereoselectivity,
however, and the diastereomers were separated by chromatog-
raphy, and each isomer was separately converted into the cor-
responding phosphorothioic acid in a Stec reaction with carbon
disulfide under basic conditions.16 The thiation proceeded with
retention of the configuration.16–19 We have adapted this approach
for our synthetic work.
aLauras, Gaustadalleen 21, N-0349, Oslo, Norway
bSchool of Pharmacy, University of Oslo, N-0315, Oslo, Norway
cDepartment of Chemistry, University of Oslo, N-0315, Oslo, Norway.
E-mail: kjell.undheim@kjemi.uio.no; Fax: +47 22855507
dThe Biotechnology Centre of Oslo, University of Oslo, N-0315, Oslo,
Norway
† Electronic supplementary information (ESI) available: Additional syn-
thetic procedures. See DOI: 10.1039/b702403g
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2 was protected at the 2ꢀ-OH substituent with a bulky silyl
group for two major reasons. Firstly, chemical interference with
other functional groups was to be avoided under the conditions
employed in the construction of the phosphoryl chloride 4 or
an equivalent. Secondly, the hydrophobic and bulky silyl group
was expected to facilitate isolation of the thioic acid after the
thiation reaction by precipitation from the complex aqueous
mixture (vide infra).
Results and discussion
The main challenge has been to achieve stereochemical control
in the construction of the new configurational center at the
phosphorus atom by way of phosphoramidate formation. All
stereoselective processes detailed below involve an amidate in-
termediate. The amidate must be formed from a primary amine
in order to have an amidate N–H proton available for abstraction
and metallation in the thiylation step (vide infra). Once the amidate
has been constructed, the P–N bond is cleaved intramolecularly
by a sulfur nucleophile with retention of the true configuration
at the phosphorus atom. There is, however, an apparent change
in the expression of the configuration at the phosphorus atom
because of the nomenclature priority rules. Deprotonation of an
amide hydrogen with a strong base provides a metallated amido-
nitrogen which reacts with carbon disulfide. A sulfur atom in the
intermediate becomes a nucleophile and a cyclisation reaction
subsequently occurs where a sulfur atom adds to the phosphorus
atom with a concurrent cleavage of the P–N bond. This process
generates a phosphorothioic acid in a stereocontrolled manner
with retention of the true configuration.
Ready variations in the nature of the 8-substituent in the
target molecules would suggest a common substrate after the
introduction of the stereochemistry at phosphorus (Scheme 1).
Convenient substrates were 8-bromo and 8-chloro derivatives. The
8-bromo derivative was available from cAMP by bromination
reactions.20 In this work bromination conditions were adapted
for larger scale operation, and the reaction was carried out in
a concentrated solution with two equivalents of sodium acetate
and neat bromine. Under these conditions, the 8-Br-cAMP was
precipitated as formed with continuous removal of the HBr
generated, thereby maintaining a constant pH in the solution.
These reaction conditions have repeatedly yielded 70–80% product
in work on a “100 g scale”. Before amidation, the bromo substrate
A practical problem may be experienced in the work with cyclic
nucleotides because of low solubility in organic solvents. The
problem was overcome for the 8-Br-cAMP which was converted
into its tri-n-butylammonium salt before silylation, since the tri-
n-butylammonium salt is soluble in dry DMF. The reactions with
tert-butyldimethylsilyl (TBDMS) chloride in DMF in the presence
of imidazole as base at 20–60 ◦C, furnished the silyl protected
compound 3. The bromine in the 8-position may be displaced
by another halide ion, and therefore the silylation was effected
under relatively mild conditions. This chloride exchange problem
could also be avoided by using tert-butyldimethylsilyl triflate
(TBDMSOTf) as a silylating reagent instead of TBDMSiCl.
Conditions for the amidation step have been varied, and in our best
procedure oxalyl chloride has been used in the presence of DMF
in THF or dichloromethane at low temperature (−60–−20 ◦C).
The actual chlorinating agent of phosphorus is probably the
Vilsmeyer reagent, (chloromethylene)dimethylammonium chlo-
ride, which is generated in situ from oxalyl chloride and DMF. The
stereochemistry of an intermediate phosphoryl chloride 4, or an
equivalent, has not been investigated. The crude halide was used
directly in the amidation step with primary amines. Benzylamine
provided the corresponding amidate. Only the (SP)-isomer 5 was
detected. Aniline provided the N-phenyl phosphoramidate (SP)-6.
Sometimes a small amount of the other diastereomer was present
but was readily removed during the purification process. The
intermediate phosphoryl chloride in the reaction may be a
Scheme 1 Reagents and reaction conditions: (i) Br2, NaOAc, H2O, rt, 24 h, (ii) TBDMS-Cl, imidazole, DMF, 60 ◦C, 20 h, (iii) TBDMS-triflate, imidazole,
CH2Cl2, rt, 20 h, (iv) (a) (COCl)2, DMF, CH2Cl2, −60 ◦C–rt, 30 min, (b) BnNH2, CH2Cl2, −60 ◦C–rt, 2.5 h, (v) (a) (COCl)2, DMF, CH2Cl2, −20 ◦C 1 h.
(b) PhNH2, rt, 3 h.
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stereochemical mixture. The main signal in 31P NMR of the
mixture was seen at −1.73 ppm. Selective formation of the amidate
with (SP)-configuration from a mixture of acid chlorides would
require some configurational equilibrations. In the (SP)-isomer, the
benzylamino or anilino group in the amidates occupies an equato-
rial position as shown by single crystal X-ray analyses (vide infra).
Any important influence of an 8-substituent in the heterocycle
on the amination selectivity at the phosphorus atom was investi-
gated. A derivative without any 8-substituent was required and was
synthesised as shown in Scheme 2. cAMP (1) was silyl-protected,
converted to its tri-n-butylammonium salt 7, and amidated as
above (Scheme 2). Only the (SP)-stereoisomer of the benzylamidate
8 was obtained. In a similar way, any stereochemical directing
effect from an 8-carbon substituent was investigated using a furyl
substrate 9. The latter was available from the 8-bromo-cAMP tri-
n-butylammonium salt 3, which was cross-coupled by Pd-catalysis
under Stille conditions in DMF where the ammonium salt is
soluble. Chlorination and amidation of substrate 9 proceeded to
furnish one stereoisomer, viz compound (SP)-10a. These experi-
ments indicate that the nature of an 8-substituent is not the main
cause of the stereoselectivity. The finding is rationalised by the
large distance between the 8-substituent and the phosphorus atom
where the reaction occurs. A common feature of all these substrates
for amination is a bulky 2ꢀ-silyloxy group which presumably will
favour a quasi-equatorial position with a flattening of the five-
membered ribose ring and subsequent conformational changes in
the annulated six-membered phosphorus ring, which eventually
lead to the (SP)-amidate with the amino group in an equatorial
position.
The amidates are neutral molecules which are soluble in several
common organic solvents. Hence these molecules are appropriate
for cross-coupling reactions in the 8-position. DMF was a good
solvent except for the preparation of the pyridinyl derivate 11c
when N-methylpyrrolidone (NMP) was a better choice for solu-
bility reasons. Cross-coupling reactions were effected by palladium
catalysis under Stille conditions and provided high yields of the
8-hetaryl substituted products 10 and 11 as shown in Scheme 3
(see the ESI†).
Scheme 3 Reagents and reaction conditions: (i) n-Bu3Sn-R, Pd(OAc)2,
PPh3; 10a DMF, 85 ◦C, 2.5 h; 11a DMF, 80 ◦C, 1 h; 11b DMF, 90 ◦C, 4 h;
11c NMP, 110 ◦C, 10 h.
The (SP)-configuration assigned to the amidates has been
ascertained by a single crystal X-ray structure analysis of the 2-
furyl derivatives 10a and 11a. The X-ray data show that both
the alkyl 10a and the aryl 11a amidates possess the same (SP)-
configuration at the phosphorus atom (Figs. 2 and 3). The
configuration in the furyl derivatives 10a and 11a is the same as in
the bromo-amidates 5 and 6 because the cross-coupling reaction
does not interfere with the configuration at the phosphorus atom.
The ORTEP plots of the crystalline structures 10a and 11a are
shown in Fig. 2 and Fig. 3, respectively.
The crystal structures of the N-benzyl and the N-phenyl ami-
dates are closely similar. The amino group occupies an equatorial
position. The bulky silyloxy group tends to reduce steric repulsions
in a quasiplanar conformation by a flattening of the ribose ring.
For the thiylation reaction in the benzyl series, n-BuLi or LDA
was used, initially at −78 ◦C. Sodium or potassium tert-butoxide
Scheme 2 Reagents and reaction conditions: (i) N-nBu3, TBDMS-Cl,
imidazole, CH2Cl2, rt, 20 h, (ii) (a) (COCl)2, DMF, CH2Cl2, −20 ◦C, 1 h,
(b) BnNH2, CH2Cl2, −20 ◦C–rt, 3 h, (iii) 2-(n-Bu3Sn)furan, Pd(OAc)2,
PPh3, DMF, 85 ◦C, 5 h, (iv) (a) (COCl)2, DMF, CH2Cl2, 0 ◦C–rt,
(b) BnNH2, CH2Cl, −20 ◦C–rt, 2 h.
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Fig. 2 ORTEP plot of compound 10a. Ellipsoids are shown at 50%
probability. For clarity only the hydrogen atoms at the stereogenic centers
are shown.
Scheme 4 Reagents and reaction conditions: (i) t-BuOK, THF, rt, CS2,
3 h, (ii) NH4F, DMF, rt, 5 d, (iii) (a) n-BuLi, THF, −78 ◦C, (b) CS2, rt, 3 h,
(iv) NH4F, 40 ◦C, 48 h.
displaced from the initial ammonium salt in situ by addition of
tri-n-butylamine (see the ESI†). The tri-n-butylammonium salts
are soluble in several organic solvents and are readily purified by
preparative chromatography. Further conversion of the purified
tri-n-butylammonium salts to sodium salts 15 were effected by
sodium hydroxide in a methanol solution.
The overall reaction sequence is remarkably chemo- and
stereoselective. Only one signal in the 31P NMR spectra was
present. cAMPS, the parent molecule, was first synthesised by
Eckstein et al.,22 and the configurational assignment of cAMPS
was first established by Stec et al.23 The 31P NMR chemical shifts
for cAMPS as sodium salt in aqueous solution were reported as
54.81 ppm, for the (SP)-, and as 56.37, for the (RP)-, isomer.24 The
value recorded for the (RP)-isomer 15d was 57.02 ppm in D2O,
and the chemical shifts for the sodium salts 15 were in the range
56.4–57.0 ppm in D2O and 58.1–58.4 ppm in MeOH-d4. The 31
P
NMR signals from the tri-n-butylammonium salts 13b, 14c and
14d (recorded in CDCl3), appeared in the range 55.7–58.2 ppm.
The resonances of the (SP)-N-benzyl amidates 5 and 10a in CDCl3
were at 8.0–8.3 ppm, which differed significantly from the (SP)-N-
phenylamidates 11a–c at 2.5–3.1 ppm. The 31P NMR shifts for the
phosphoric acid derivatives as tri-n-butylammonium salts 3, 7 and
9 were in the range −1.1–−2.0 ppm. Thus, the phosphorus NMR
shifts are useful diagnostic tools for structure assignments. The
31P NMR shift for (RP)-cAMPS as sodium salt in D2O is found
at a lower chemical field than for its (SP)-cAMPS diastereomer.24
In the correlation of the configuration at the phosphorus atom
in the thioic acids by 31P NMR shifts, spectroscopic data from
both stereoisomers of a phosphorothioic acid would be required.
A new synthetic approach had to be devised for making both
diastereomers available (Scheme 6). This pathway is inferior
to the one outlined above for the preparation of pure (RP)-
isomers, but from the mixture of stereoisomers prepared, as in
Scheme 6, the (SP)-isomer became available. The substrate was
Fig. 3 ORTEP plot of compound 11a. Ellipsoids are shown at 50%
probability. For clarity only the hydrogen atoms at the stereogenic centers
are shown.
was used for proton abstraction in the phenyl series 11 at room
temperature. The 6-amino group in the purine did not appear
to cause any adverse reactions. The thiations of the amidates by
means of carbon disulfide (the Stec reaction),21 were stereospecific.
The reaction sequence for the 8-furyl N-benzyl- and N-phenyl-
amidates 10a and 11a is shown in Scheme 4. The thiylation
product 12a is the same from both substrates. The silyl function
was removed by ammonium fluoride, and the product was
isolated as the ammonium salt 13a. More conveniently, the
phosphorothioic acid was isolated as its tri-n-butylammonium
salt. After desilylation as shown in Scheme 5, the ammonia was
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Scheme 5 Reagents and reaction conditions: (i) t-BuOK, THF, rt, CS2, 3 h, (ii) (a) n-BuLi, THF, −78 ◦C, 10 min, (b) CS2, rt, 3 h, (iii) (a) NH4F, DMF,
rt, 5 d, (b) n-Bu3N, DMF, rt, (iv) (a) NaOH, MeOH, rt, (b) hexane.
product 12 from a complex aqueous mixture in the work up
of the reaction. The purity of the product, thus obtained, was
often sufficient for direct silyl deprotection with fluorides such as
ammonium fluoride. With ammonium fluoride in DMF at room
temperature, the reaction was slow and required 3–5 days. With
cesium fluoride, however, the reaction in the cold required only a
few hours, but the product was less pure.
Conclusion
A method for stereocontrolled preparation of 8-carbon substi-
tuted (RP)-adenosine-3ꢀ,5ꢀ-cyclic phosphorothioic acids has be-
come available. Stereochemistry at phosphorus was introduced
by stereoselective amidation. Phosphoramidates with the (SP)-
configuration have been converted by thiylation into phosphoroth-
ioic acids with the (RP)-configuration. cAMP has been efficiently
brominated to provide 8-bromo-cAMP which, in a reaction
sequence, has been cross-coupled under Pd-catalysis to provide 8-
Scheme 6 Reagents and reaction conditions: (i) PSCl3, pyridine, −8 ◦C,
hetaryl derivatives as substrates for 8-hetaryl-cAMPS derivatives.
90 min, (ii) (a) NaOH, MeCN–H2O (50%), rt, (b) solid CO2.
The cAMP substrates were 2Oꢀ-protected by a bulky silyl group.
The nature of the purine 8-substituent did not exert any marked
effect on the stereoselectivity in the amidation.
8-(2-furyl)adenosine25 16 which was reacted with thiophosphoryl
chloride in dry pyridine to furnish a thiophosphoryl intermediate
17. For this reaction to proceed well, it is essential that both the
solvent and reactants have been well dried. Base treatment of the
phosphorylated product in the cold provided the crude sodium salt
of a diastereomeric mixture of the (RP)- and (SP)-isomers 15 in the
ratio 1 : 3. Thus this approach favours preparation of the (SP)-
isomer. The 31P NMR shift values of the 8-furyl diastereomers
as sodium salts in D2O were at 55.45 ppm for the (SP)-isomer
15aa and at 56.20 ppm for the (RP)-isomer 15a as compared with
the values 54.81 ppm for (SP)-15d and 56.37 for (RP)-15d.24 Thus
the (SP)-configuration of the amidates, as determined by the X-
ray analysis, can be correlated with the (RP)-configuration in the
phosphorothioic acid series by phosphorus NMR shift values.
The bulky silyl protecting group is important for the isolation
of the first formed phosphorothioic acid 12 because it renders the
product insoluble and assists in the precipitation of the thiated
Experimental
1H NMR spectra were recorded in CDCl3 or DMSO at 200, 300
and 500 MHz with Bruker DPX 200, 300 and 500 instruments.
The 13C NMR spectra were recorded at 75, 100 and 150 MHz.
Chemical shifts are reported in ppm using CHCl3 (7.24 ppm) and
1
CDCl3 (77 ppm) as references, and in DMSO, 2.49 ppm in H
NMR and 39.5 ppm in 13C NMR. The 31P spectra were recorded
in CDCl3, MeOH-d4 or D2O at 81 MHz or 121 MHz with a
Bruker DPX 200 or 300 instrument with 85% H3PO4 as an external
reference. Mass spectra were recorded at 70 eV with a Fisons VG
Prospectrometer. The spectra are presented as m/z (% relative
intensity). Electrospray spectra were obtained with a Micromass
QTOF 2 W spectrometer with electrospray ionisation quadrupole
time of flight.
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THF was distilled from sodium–benzophenone. Dichloro-
methane and tri-n-butylamine were distilled from calcium hydride.
Flash chromatography on silica gel was carried out on Merck
kieselgel 60 (230–400).
room temperature. The mixture was stirred under argon at 60 ◦C
for 20 h. The solvent was removed at reduced pressure, the residual
material was suspended in water (150 mL), and was stirred for
30 min to hydrolyse any silylated phosphoric acid. Then 1 M HBr
(ca. 12 mL to pH 2–3) was added to the filtrate. The precipitate
was filtered off, washed with water, and dried under vacuum. The
acid was suspended in MeOH (80 mL) and n-Bu3N (5 mL) was
added. The mixture was stirred at room temperature until a clear
solution was obtained (30 min). The solvent was distilled off, and
the residual salt product was dried under vacuum over P2O5 before
being used as such in the subsequent reaction, yield 6.60 g (78%) of
a white solid. HRMS (Electrospray, TOF ES−): M 520.0427. Calc.
for C16H24BrN5O6PSi: 520.0422. 31P NMR (CDCl3, 81 MHz): d
X-Ray crystallographic analysis for compounds 10a and 11a‡
X-Ray data were collected on a Siemens SMART CCD
diffractometer26 using graphite monochromated MoKa radiation
◦
˚
(k = 0.71073 A). Data collection method: x-scan, range 0.6 ,
crystal to detector distance 5 cm. Data reduction and cell determi-
nation were carried out with the SAINT and XPREP programs.26
Absorption corrections were applied by the use of the SADABS
program.27 The structures were determined and refined using the
SHELXTL program package.28 The non-hydrogen atoms were
refined with anisotropic thermal parameters, hydrogen atoms were
located from difference Fourier maps and refined with isotropic
thermal parameters.
1
−1.36; H NMR (DMSO-d6, 300 MHz): d 0.01 (3H, s, Si-CH3),
0.03 (3H, s, Si-CH3), 0.83 (9H, s, Si-tBu), 0.87 (9H, t), 1.28 (6H,
m), 1.53 (6H, m), 2.79 (6H, m), 3.90 (2H, m), 4.11 (1H, m), 5.02
(2H, m), 7.53 (2H, s, NH2), 8.15 (1H, s, H-2); 13C NMR (DMSO-
d6, 75 MHz): d −5.3, −4.7, 13.6, 18.0, 19.6, 25.6, 25.7, 51.9, 65.4,
72.3, 72.4, 76.2, 94.3, 119.2, 126.5, 150.0, 153.2, 155.0.
Crystal data for C27H35N6O6PSi (10a). M = 598.67, ortho-
˚
−1
rhombic, P212121, a = 10.853(1), b = 13.388(1), c = 20.924(1) A,
Method (ii). TBDMS-triflate (4.75 g, 4.13 mL, 18 mmol) was
added to a suspension of 8-Br-cAMP (2) as tri-n-butylammonium
salt (5.3 g, 8.9 mmol) and imidazole (1.72 g, 27 mmol) in CH2Cl2
(40 mL) at room temperature, and the mixture stirred under argon
for 20 h. The solvent was removed, the residual material suspended
in water (120 mL), stirred for 30 min to hydrolyse any silylated
phosphoric acid. Then 1 M HBr (ca. 10 mL) was added to the
filtrate to pH 2–3. The precipitate was filtered off, washed with
water, and dried at reduced pressure. The acid was suspended in
MeOH (60 mL) and n-Bu3N (4 mL) was added. The mixture was
stirred at room temperature until a clear solution was obtained
(30 min), and the solvent distilled off. The residual product was
dried at reduced pressure over P2O5. The crude product was used
as such in the subsequent reaction, yield 4.93 g (78%) of a white
solid salt. Physical data as above.
3
−3
˚
V = 3040.3(1) A , Z = 4, Dx = 1.308 Mg m , l = 0.180 mm ,
T = 120(2) K, measured 57613 reflections in 2h range 3.6–72.6◦,
Rint = 0.033. 510 parameters refined against 14434 F2, R = 0.032
for Io > 2r(Io) and 0.042 for all data.
Crystal data for C26H33N6O6PSi (11a). M = 584.64, ortho-
˚
−1
rhombic, P212121, a = 10.913(1), b = 13.523(1), c = 20.290(1) A,
3
−3
˚
V = 2994.3(1) A , Z = 4, Dx = 1.297 Mg m , l = 0.181 mm ,
T = 105(2)K, measured 39032 reflections in 2h range 3.6–56.6◦,
Rint = 0.031. 493 parameters refined against 7452 F2, R1 = 0.024
for Io > 2r(Io) and 0.025 for all data.
8-Bromoadenosine-3ꢀ,5ꢀ-cyclic phosphoric acid (2)
Bromine (15.4 mL, 0.30 mol) was added with stirring to a solution
of cAMP (98.8 g, 0.30 mol) and sodium acetate trihydrate (81.6 g,
0.60 mol) in water (1.5 L) over 1 h at room temperature. Sodium
sulfite was added slowly after 24 h until disappearance of the dark
red colour. The precipitate was collected by filtration, the solid
washed with water, with 2-propanol and diethyl ether, and the
product was dried at reduced pressure. The product was dispersed
in water (500 mL) and dissolved by slow addition of sodium
bicarbonate (1 eq.). When all the material had dissolved, small
portions of sodium sulfite were added to remove the dark red
colour of the solution. Precipitation of the product was effected
by dropwise addition of 1.0 M hydrobromic acid under vigorous
stirring. The precipitate was collected, washed with water, 2-
propanol, and diethyl ether, and the bright yellow powder dried
under high vacuum, yield 92.0 g (76%). 1H NMR analysis was in
accordance with the literature data.20
(SP)-8-Bromoadenosine-2ꢀO-(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic
N-benzylphosphoramidate (5)
A solution of 8-bromadenosine-2ꢀO-(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-
cyclic phosphoric acid tri-n-butylammonium salt (3) (7.070 g,
10.0 mmol) in CH2Cl2 (70 mL) was added dropwise to a solution
of oxalyl chloride (5.5 mL, 11.0 mmol, 2 M in CH2Cl2) and DMF
(0.58 mL, 3.6 mmol) in CH2Cl2 (70 mL) at −60 ◦C. Benzylamine
(40 mmol, 4.37 mL), (dried over calcium hydride), was added
after 30 min, and the mixture was stirred at −60 ◦C for 10 min and
at room temperature for 2 h, diluted with CHCl3 (100 mL) and
washed with saturated NaHCO3(aq.) (2 × 50 mL). The organic
phase was dried (MgSO4), the solvent was distilled off and the
residue was purified by flash chromatography on silica gel using
CH2Cl2–MeOH (3 : 97 and 5 : 95); yield 3.00 g (49%) of a white
solid. The product was dissolved in CH2Cl2 and reprecipitated as
a white powder on slow addition of hexane, mp slow decomp.
190–200 ◦C (brown–black remains no mp <300 ◦C). Electrospray
(TOF MS ES+): 611.2/613.2; 31P NMR (CDCl3, 121 MHz): d 8.29;
1H NMR (CDCl3, 300 MHz): d 0.011 (3H, s, Si-CH3), 0.014 (3H, s,
Si-CH3), 0.82 (9H, s, Si-tBu), 3.81–3.90, (1H, m, NH), 4.12–4.21
(3H, m), 4.39–4.68 (3H, m), 4.97 (1H, d, J 5.1 Hz), 5.54–5.60 (1H,
m), 5.88 (1H, s), 6.50 (2H, br s, NH2), 7.19–7.33 (5H, m), 8.04
(1H, s, H-2); 13C NMR (CDCl3, 75 MHz): d −5.1, −4.7, 18.1,
8-Bromo-2ꢀO-(tert-butyldimethylsilyl)adenosine-3ꢀ,5ꢀ-cyclic
phosphoric acid tri-n-butylammonium salt (3)
Method (i). TBDMS-Cl (2.72 g, 18 mmol) was added to a
solution of 8-Br-cAMP (2) as tri-n-butylammonium salt (7.0 g,
11.8 mmol) and imidazole (2.45 g, 36 mmol) in DMF (30 mL) at
‡ CCDC reference numbers 625282 (11a) and 625283 (10a). For crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/b702403g
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(SP)-2ꢀO-(tert-Butyldimethylsilyl)adenosine-3ꢀ,5ꢀ-cyclic
25.6, 45.3, 68.2 (Jp 6.9 Hz), 71.3 (Jp 4.2 Hz), 73.0 (Jp 8.3 Hz),
76.3 (Jp 3.6 Hz), 94.7, 120.1, 127.0, 127.4, 128.5, 138.7, 139.7(Jp
6.5 Hz), 150.4, 153.5, 154.4.
N-benzylphosphoramidate (8)
Dry DMF (0.385 mL, 5.00 mmol) in dry CH2Cl2 (40 mL)
was placed under an atmosphere of argon gas and cooled
to 0 ◦C before oxalyl chloride (4 mL, 2 M in CH2Cl2,
8.14 mmol) was added slowly. The cooling bath was removed
and the suspension left to stir at room temperature for 30 min.
The reaction mixture was cooled to −20 ◦C, and a solution
of 2ꢀO-(tert-butyldimethylsilyl)adenosine-3ꢀ,5ꢀ-cyclic phosphoric
acid tri-n-butylammonium salt (7) (4.650 g, 7.4 mmol) in dry
dichloromethane (30 ml) was added through a Teflon tube. The
mixture was stirred at this temperature for 1 h, and benzylamine
(3.2 mL, 29 mmol) (dried over CaH2) was added. The cooling
bath was removed after 1 h and stirring was continued at room
temperature for 2 h before the turbid reaction mixture was
filtered. Dichloromethane (120 mL) was added to the filtrate before
extraction with cold, saturated sodium hydrogen carbonate (2 ×
50 mL). The organic phase was dried (MgSO4), the solvent was
removed at reduced pressure, and the residual material subjected to
flash chromatography on silica gel using 10% methanol in CH2Cl2;
yield 1.92 g (49%) of a white solid. The product was dissolved in
CH2Cl2 and reprecipitated as a white powder on slow addition of
hexane, m◦p slow decomp. 200–210 ◦C (brown–black remains, no
mp <300 C). HRMS (Electrospray, TOF ES+): 533.2076. Calc.
for C23H33N6O5PSi + H+: 533.2092. 31P (CDCl3, 121 MHz): d 7.92;
1H NMR (CDCl3, 200 MHz): d −0.05 and 0.0 (6H, 2s, 2 × Si-
Me), 0.76 (9H, s, Si-tBu), 3.97–4.03 (2H, m), 4.17–4.22 (2H, m),
4.35–4.56 (2H, m), 4.64 (1H, d, J 5.1 Hz), 4.96–5.08 (1H, m), 5.83
(2H, br s, NH2), 5.86 (1H, s), 7.13–7.20 (5H, m), 7.88 (1H, s), 8.12
(1H, s, H-2); 13C NMR (MeOH, 75 Mz): d −1.65, −1.48, 17.3,
21.5, 48.5, 69.4, 72.0, 74.55, 77.5, 96.6, 122.8, 130.5, 130.9, 131.9,
142.1, 142.4, 152.4, 156.7, 159.1.
(SP)-8-Bromoadenosine-2ꢀO-(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic
N-phenylphosphoramidate (6)
A solution of dry DMF (0.289 g, 3.96 mmol) in dry THF
(20 mL) under argon gas was cooled to 0 ◦C and oxalyl chloride
in dichloromethane (2 mL, 2 M, 4 mmol) was added slowly.
The cooling bath was removed and the suspension was left
with stirring at room temperature for 30 min. The reaction
mixture was cooled to −20 ◦C and added to a solution of
tri-n-butylammonium 8-bromoadenosine-2ꢀO-TBDMS-3,5-cyclic
monophosphate (3) (2.55 g, 3.60 mmol) in dry dichloromethane
(8 mL). The mixture was stirred at this temperature for 1 h,
and was allowed to reach room temperature before dry aniline
(3.35 g, 36 mmol) was added. The aniline had previously been
dried (calcium hydride). After 3 h, the turbid reaction mixture
was diluted to 100 mL with dichloromethane and washed with
cold, saturated sodium hydrogen carbonate (3 × 25 mL). The
organic phase was dried (MgSO4), the solvent was removed at
reduced pressure, and the residual material was added slowly with
vigorous stirring to cyclohexane (100 mL). The precipitate was
dried and subjected to flash chromatography on silica gel using
7% methanol in dichloromethane, yield 1.46 g (68%). 31P NMR
(DMSO-d6, 81 MHz): d 2.28; 1H NMR (DMSO-d6, 300 MHz): d
−0.09 (3H, s, Si-CH3), 0.02 (3H, s, Si-CH3), 0.79 (9H, s, Si-tBu),
4.4 (2H, m), 4.65 (1H, m), 5.12 (1H, d), 5.52 (1H, m), 5.92 (1H,
s), 6.93 (1H, t), 7.10 (2H, d), 7.20 (2H, t), 7.56 (2H, s, NH2),
8.23 (1H, s, H-2), 8.55 (1H, d, Ar-NH); 13C NMR (DMSO-d6,
75 MHz): d −5.2–5.0, 17.8, 24.9, 68.2, 70.4, 72.3, 76.1, 92.9, 117.7,
118.5, 121.8, 128.9, 135.9, 139.5, 149.7, 153.4, 155.1.
2ꢀO-(tert-Butyldimethylsilyl)adenosine-3ꢀ,5ꢀ-cyclic phosphoric acid
tri-n-butylammonium salt (7)
8-(2-Furyl)adenosine-2ꢀO-(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic
phosphoric acid tri-n-butylammonium salt (9)
TBDMS-triflate (8.04 mL, 35 mmol) was added to a suspension
of cAMP (1) as tri-n-butylammonium salt (8.99 g, 17.5 mmol)
and imidazole (3.57 g, 52.5 mmol) in CH2Cl2 (60 mL) at room
temperature. The mixture was stirred at room temperature under
argon for 20 h. The solvent was removed, and the residual material
suspended in water (120 mL) and stirred for 30 min to hydrolyse
any silylated phosphoric acid. Then 1 M HBr was added to the
filtrate to pH 2–3. The precipitate was filtered off, and was washed
with water and dried under vacuum. The acid was suspended in
MeOH (60 mL) and n-Bu3N (5 mL) was added. The mixture was
stirred at room temperature until a clear solution was obtained
(30 min). The solvent was distilled off, and the residual product
was dried under vacuum over P2O5 before being used as such in
the subsequent reaction, yield 8.24 g (75%) of a white salt.
31P (CDCl3, 121 MHz): d −2.01; 1H NMR (CDCl3, 200 MHz):
d 0.01 (6H, s, 2 × Si-Me), 0.79 (9H, s, Si-tBu), 0.79–0.85 (9H, t),
1.16–134 (6H, m), 1.49–1.65 (6H, m), 2.83–2.91 (6H, m), 4.14–4.27
(3H, m), 4.50–4.61 (2H, m, ), 5.89 (1H, s), 6.47 (2H, br s, NH2),
7.81 (1H, s, ), 8.16 (1H, s, H-2); 13C NMR (CDCl3, 50 Mz): d −4.6,
−4.2, 13.9, 18,6, 20.3, 25.7, 26.1, 52.7, 67.5, 72.6, 74.9, 76.2, 93.3,
118.8, 138.6, 149.5, 153.5, 156.2.
A solution of Pd(OAc)2 (0.170 g, 0.76 mmol) and PPh3 (0.420 g,
1.6 mmol) in dry, degassed DMF (20 mL) was stirred at
50 ◦C until the solution had turned dark red. A solution of
8-bromoadenosine-2ꢀO-(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic phos-
phoric acid tri-n-butylammonium salt (3) (2.700 g, 3.8 mmol) in
DMF (10 mL) together with 2-(tri-n-butylstannyl)furan (1.7 g,
4.77 mmol) was added. The reaction mixture was stirred at 90 ◦C
for 5 h. The DMF was removed at reduced pressure, and the
residual material was subjected to flash chromatography on silica
gel using CH2Cl2–MeOH–NBu3 (100 : 7.5 : 1). The coupled
product was isolated as a white solid material which contained
traces of organotin residues which were effectively removed by
dissolution in CH2Cl2 and precipitation by hexane, yield 1.8 g
(72%). HRMS (Electrospray, TOF ES−): 508.1407. Calc. for
1
[C20H27N5O7PSi]−: 508.1422. 31P (CDCl3, 121 MHz): d −1.1; H
NMR (CDCl3, 200 MHz): d −0.05 and 0.05 (6H, 2 s, 2 × Si-Me),
0.77 (9H, s, Si-tBu), 0.88–0.95 (9H, t), 1.29–1.40 (6H, m), 1.53–
1.71 (6H, m), 2.87–2.96 (6H, m), 4.18–4.34 (3H, m, ), 5.08 (1H,
d, J 5.1 Hz), 5.39–5.44 (1H, m), 6.07 (2H, br s, NH2), 6.19 (1H,
s), 6.55–6.58 (1H, m), 7.08–7.10 (1H, m), 7.61–7.62 (1H, m), 8.28
(1H, s, H-2); 13C NMR (CDCl3, 50 Mz): −5.3, −4.3, 13.6, 18.2,
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20.1, 25.1, 25.7, 51.7, 67.5, 72.6, 74.9, 76.2, 94.4, 111.9, 113.9,
119.7, 142.1, 143.4, 144.9, 150.5, 153.1, 155.2.
solid. HRMS (Electrospray, TOF ES+): M 585.2023. Calc. for
C26H34N6O6PSi: 585.2041. 31P NMR (CDCl3, 81 MHz): d 3.06;
1H NMR (DMSO-d6, 300 MHz): d −0.14 (3H, s, Si-CH3), −0.11
(3H, s, Si-CH3), 0.70 (9H, s, Si-tBu), 4.4–4.5 (2H), m, 4.65 (1H,
dm), 5.19 (1H, d), 5.65 (1H, m), 6.31 (1H, s), 6.77 (1H, dd), 6.93
(1H, t), 7.1–7.2 (5H, m), 7.58 (2H, br s), 8.00 (1H, d), 8.27 (1H,
s), 8.56 (1H, d); 13C NMR (DMSO-d6, 75 MHz): d −5.6, −5.0,
17.7, 25.3, 68.3, 70.2, 72.4, 76.3, 93.6, 112.3, 113.9, 118.45, 121.8,
128.9, 139.6, 140.3, 143.2, 145.6, 149.8, 153.4, 156.1.
(SP)-2ꢀO-(tert-Butyldimethylsilyl)-8-(2-furyl)adenosine-3ꢀ,5ꢀ-cyclic
N-benzyl phosphoramidate (10a)
(i) By amidation of the cyclic phosphoric acid tri-n-butylammo-
nium salt 9. Dry DMF (0.1 mL, 1.3 mmol) in dry CH2Cl2 (20 mL)
was placed under an atmosphere of argon gas and cooled to
◦
0 C before oxalyl chloride (1 mL, 2 M in CH2Cl2, 2 mmol) was
added slowly. The cooling bath was removed and the suspension
stirred at room temperature for 30 min, cooled to −20 ◦C and
a solution of 8-(2-furyl)adenosine-2ꢀO-(tert-butyldimethylsilyl)-
3ꢀ,5ꢀ-cyclic phosphoric acid tri-n-butylammonium salt (9) (1.2 g,
1.75 mmol) in dry CH2Cl2 (15 mL) was added through a Teflon
tube. The mixture was stirred at this temperature for 1 h, dry
(CaH2) benzylamine (0.76 mL, 7 mmol) added, and the mixture
was stirred at room temperature for 2 h when the turbid reaction
mixture was filtered. Then CH2Cl2 (120 mL) was added to
the filtrate, the solution shaken and the solvent removed at
reduced pressure. The residual material was subjected to flash
chromatography on silica gel using 4% methanol in CH2Cl2; yield
0.745 g (71%) of a white solid. The product was dissolved in
CH2Cl2 and reprecipitated as a white◦powder on slow addition
of hexane, mp◦slow decomp. 190–200 C (brown–black remains,
no mp <300 C). Electrospray MS (TOF MS ES+): 599.2; 31P
(CDCl3, 121 MHz): d 7.94; 1H NMR (CDCl3): d −0.05 and 0.02
(6H, 2s, 2 × CH3), 0.78 (9H, s, C(CH3)3), 3.61–3.72 (1H, m, NH),
4.13–4.23 (3H, m), 4.35–4.59 (2H, m), 5.11 (1H, d, J 5.2 Hz),
5.65–5.73 (1H, m), 6.07 (2H, br s, NH2), 6.30 (1H, s), 6.58–6.61
(1H, m), 7.10–7.13 (1H, m), 7.28–7.33 (5H, m), 7.61–7.62 (1H, m),
8.26 (1H, s, H-2); 13C NMR (CDCl3, 75 Mz): d −5.3, −4.6, 18.05,
25.5, 45.4, 68.4, 71.3, 73.1, 77.2, 94.2, 112.1, 114.1, 119.7, 127.1,
127.5, 128.6, 138.7, 141.8, 143.5, 144.9, 150.3, 153.2, 155.2.
(SP)-2ꢀO-(tert-Butyldimethylsilyl)-8-(N-methylpyrrol-
2-yl)adenosine-3ꢀ,5ꢀ-cyclic N-phenylphosphoramidate (11b)
Compound 11b was prepared as above from (SP)-8-bromo-
adenosine-2ꢀO-(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic-N-phenylphos-
phoramidate (6) in 70% yield. HRMS (electrospray, TOF
ES+): M + H 598.2339. Calc. for [C27H36N7O5PSi + H+]: 598.2357.
31P NMR (CDCl3), 121 MHz): d 2.54 ppm.
(SP)-2ꢀO-(tert-Butyldimethylsilyl)-8-(3-pyridinyl)adenosine-3ꢀ,5ꢀ-
cyclic N-phenylphosphoramidate (11c)
A solution of Pd(OAc)2 (37 mg, 0.166 mmol) and PPh3 (91 mg,
0.348 mmol) in NMP (4 mL) under argon was stirred at 50 ◦C
for 30 min when the solution had turned dark red. A solu-
tion of (SP)-8-bromoadenosine-2ꢀO-(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-
cyclic N-phenylphosphoramidate (6) (0.500 g, 0.83 mmol) in NMP
(2 mL) and 3-(tri-n-butylstannyl)pyridine (0.610 g, 1.66 mmol) was
added. The reaction mixture was stirred at 110 ◦C for 10 h before
the NMP was removed at reduced pressure. The residual material
subjected to flash chromatography on silica gel using 7.5% MeOH
in CH2Cl2. The product was a white solid contaminated with traces
of organotin residues which were removed by dissolution of the
coupling product in CH2Cl2 and reprecipitation by hexane, yield
0.280 g (57%); HRMS (electrospray, TOF ES+): M + H 596.2211.
Calc. for [C27H34N7O5PSi + H+]: 596.2201. 31P NMR (CDCl3,
(ii) By Pd-catalysed cross-coupling from the benzylphospho-
ramidate 5. A solution of Pd(OAc)2 (60 mg, 0.27 mmol) and
PPh3 (142 mg, 0.54 mmol) in DMF (8 mL) under argon was
stirred at 50 ◦C for 15 min before 2-(tri-n-butylstannyl)furan
(0.63 ml, 2.0 mmol) was added. Subsequently, a solution of (SP)-8-
bromoadenosine-2ꢀO-(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic N-ben-
zylphosphoramidate (5) (800 mg, 1.3 mmol) in DMF (10 mL)
was added. The mixture was stirred at 85 ◦C for 2.5 h. The
solvent was evaporated off, and the residue was purified by flash
chromatography on silica gel using CH2Cl2–MeOH (3 : 97 and 5 :
95); yield 636 mg (80%) of a white solid.
1
121 MHz): d 2.73 ppm, H NMR (CDCl3, 300 MHz): d −0.16
(3H, s, CH3), −0.15 (3H, s, CH3), 0.60 (9H, s, C(CH3)3), 4.30–4.43
(1H, m, H-4ꢀ), 4.60–4.68 (2H, m, OCH2), 5.15 (1H, d, J 5.2 Hz,
H-2ꢀ), 5.69 (1H, s, H-1ꢀ), 5.75–5.82 (1H, m, H-3ꢀ), 6.37 (2H, br s,
NH2), 6.58 (1H, d, J 9.2 Hz, NH), 6.99–7.10 (3H, m, 3 × H-Ph),
7.17–7.24 (2H, t, J 7.4 Hz, 2 × H-Ph), 7.42–7.48 (1H, m, H-pyr),
8.02–8.06 (1H, m, H-pyr), 8.37 (1H, s, H-2), 8.76–8.79 (1H, m, H-
pyr), 8.97 (1H, d, J 1.7 Hz, H-pyr); 13C NMR (CDCl3, 75 MHz):
d −5.5 and −4.8 (2 × CH3), 18.0 (Si-C), 25.4 (3 × CH3), 68.9 (d, J
6.8 Hz, OCH2), 71.3 (d, J 4.5 Hz, CH-4ꢀ), 73.3 (d. J 8.8 Hz, CH-2ꢀ),
77.5 (d, J 3.8 Hz, CH-3ꢀ), 94.2 (CH-1ꢀ), 119.4, 119.5, 119.6, 122.9,
123.5, 125.0, 129.1, 129.1, 136.8, 138.5, 148.1, 149.8, 150.3, 151.4,
153.5, 155.9.
(SP)-8-(2-Furyl)adenosine-2ꢀO-(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic
N-phenylphosphoramidate (11a)
A solution of Pd(OAc)2 (99 mg, 0.37 mmol) and PPh3 (253 mg,
0.81 mmol) in DMF (15 mL) under argon was stirred at 50 ◦C for
15 min when the mixture had become reddish brown. A
solution of tri-n-butyl(2-furyl)stannane (1.4 mL, 4.4 mmol) was
added, followed by a solution of (SP)-8-bromadenosine-2ꢀO-
(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic N-phenylphosphoramidate
(6) (2.2 g, 3.7 mmol) in DMF (10 mL). The mixture was
stirred at 80 ◦C for 1 h. The solvent was evaporated, and the
residue subjected to flash chromatography on silica gel using
CH2Cl2–MeOH (3 : 97 and 5 : 95); yield 1.93 g (90%) of a white
(RP)-2ꢀO-(tert-Butyldimethylsilyl)-8-(2-furyl)adenosine-3ꢀ,5ꢀ-cyclic
phosphorothioic acid (12a)
A 1.0 M solution of t-BuOK in THF (3.6 mL, 3.6 mmol)
was added to a solution of (SP)-2ꢀO-(tert-butyldimethylsilyl)-
8-(2-furyl)adenosine-3ꢀ,5ꢀ-cyclic N-phenylphosphoramidate (11a)
(1.51 g, 2.6 mmol) in THF (30 mL) at room temperature. The
mixture was stirred at this temperature for 1 h before CS2 (0.47 mL,
7.8 mmol) was added. The mixture was stirred for another 3 h
at room temperature, the volume of the solvent was reduced to
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about 10 mL, and hexane (90 mL) was added. The precipitate was
collected by filtration, suspended in water (55 mL), and 1.2 M
HCl (9 mL) was added. The product was collected by filtration,
washed with water and dried, yield 1.23 g (91%) of a light tan
solid. HRMS (Electrospray, TOF ES−): M 526.1326. Calc. for
C20H29N5O6PSSi: 526.1339. 31P NMR (DMSO-d6, 81 MHz): d
temperature for 3 h before the solvent was partially removed at
reduced pressure. Addition of hexane to the residual solution
precipitated a solid, which was filtered off and dissolved in water
(10 mL). Then 1 M HBr was bubbled into the solution at 0 ◦C
to reach pH 2–3. The resulting (RP)-8-(2-furyl)adenosine-2ꢀO-
(tert-butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic phosphorothioic acid 12a was
collected by filtration and dried overnight at high vacuum.
The dried phosphorothioic acid 12a (1.050 g, 2.0 mmol) was
dissolved in DMF (10 mL). Then NH4F (0.520 g, 14 mmol) was
added, and the mixture was stirred at room temperature for 5 d and
filtered. Then n-Bu3N (0.740 g, 4.0 mmol) was added until a clear
solution resulted, and the solvent was removed at reduced pressure.
The residue was washed with hexane to remove the excess of n-
Bu3N before being subjected to flash chromatography on silica gel
using CH2Cl2–CH3OH–n-Bu3N (100 : 5 : 1). The ammonium salt,
which contained some n-Bu3N, was further purified by dissolution
in CH2Cl2 and reprecipitation by hexane addition, yield 0.656 g
(40% from 10a) HRMS (Electrospray, TOF ES−): M 410.0332.
Calc. for [C14H13N5O6PS]−: 526.0339. 31P (CDCl3, 121 MHz) d
56.66; 1H NMR (CDCl3, 200 MHz): d 0.93 (9H, t, J 7.2 Hz), 1.35–
1.48 (6H, m), 1.69–1.71 (6H, m), 2.91–3.04 (6H, m), 4.35–4.53
(3H, m), 5.15 (1H, d, J 5.2 Hz), 5.6–5.79 (1H, m), 5.81 (2H, br
s), 6.24 (1H, s), 6.59–6.63 (1H, m), 7.11–7.13 (1H, m, ), 7.62–7.63
(1H, m), 8.21 (1H, s, H-2); 13C NMR (CDCl3, 75 MHz): d 13.6,
21.1, 25.2, 51.9, 67.2, 71.7, 72.5, 77.5, 94.3, 113.2, 115.3, 120.1,
143.4, 144.4, 146.8, 151.4, 154.0, 156.9.
1
58.4; H NMR (DMSO-d6, 300 MHz): d −0.03 (3H, s, Si-CH3),
0.04 (3H, s, Si-CH3), 0.79 (9H, s, Si-t-Bu), 4.0–4.5 (3H, m), 5.06
(1H, d), 5.34 (1H, m), 6.21 (1H, s), 6.79 (1H, q), 7.18 (1H, d),
8.02 (1H, s), 8.2 (2H, br s), 8.33 (1H, s); 13C NMR (DMSO-d6,
75 MHz): d −5.4, −4.6, 17.9, 25.5, 67.7, 71.1, 72.8, 75.9, 93.2,
112.4, 114.4, 119.1, 141.3, 142.8, 146.0, 149.4, 150.2, 153.7
(RP)-8-(2-Furyl)adenosine-3ꢀ,5ꢀ-cyclic phosphorothioic acid
ammonium salt (13a)
(i) Prepared from the phosphorothioic acid 12a. A solution
of (RP)-2ꢀO-(tert-butyldimethylsilyl)-8-(2-furyl)adenosine-3ꢀ,5ꢀ-
cyclic phosphorothioic acid (12a) (1.15 g, 2.7 mmol) and NH4F
(225 mg, 6.0 mmol) in DMF (10 mL) under argon was stirred
at room temperature for 5 d. Subsequently, TMSOMe (1 mL)
was added and the stirring was continued for 24 h. The solvent
was distilled off at reduced pressure, the crude product was
suspended in MeOH (10 mL), and Et2O (80 mL) added. The
light tan-coloured solid was filtered off and dried, yield 847 mg
(90%). HRMS (Electrospray, TOF ES−): M 410.0330. Calc. for
C14H13N5O6PS: 410.0329. 31P NMR (DMSO-d6, 81 MHz): d 54.3;
1H NMR (DMSO-d6, 300 MHz): d 4.0–4.2 (4H, m), 5.03 (1H, d),
5.14 (1H, m), 6.02 (1H, s), 6.76 (1H, dd), 7.13 (1H, d), 8.01 (1H,
d), 8.21 (1H, s); 13C NMR (DMSO-d6, 75 MHz): d 66.15, 75.3,
75.4, 76.9, 92.8, 112.3, 113.8, 118.9, 141.2, 143.1, 145.8, 149.8,
152.7, 155.6.
(RP)-8-(N-Methylpyrrol-2-yl)adenosine-3ꢀ,5ꢀ-cyclic
phosphorothioic acid tri-n-butylammonium salt (14b)
A mixture of (SP)-2ꢀO-(tert-butyldimethylsilyl)-8-(N-methylpyrrol-
2-yl)adenosine-3ꢀ,5ꢀ-cyclic
N-phenylphosphoramidate
(11b)
(0.300 g, 0.5 mmol) in dry THF (6 mL) and potassium tert-
butoxide (0.62 mL, 0.62 mmol, 1 M in THF) was stirred under
argon at room temperature for 1 h before carbon disulfide
(0.09 ml, 1.5 mmol) was added. The reaction mixture was stirred
at room temperature for 3 h. Most of the solvent was distilled off
at reduced pressure. Addition of hexane gave a solid precipitate
which was dissolved in water (9 mL), and 1.2 M HCl (1.25 mL)
was added at 0 ◦C. The precipitate was the silylated (RP)-8-(N-
methylpyrrol-2-yl)adenosine-3ꢀ,5ꢀ-cyclic phosphorothioic acid
(12b). The product was well dried in vacuo before the solid
(0.170 g, 0.3 mmol) was dissolved in DMF (1.5 mL) under
argon, and ammonium fluoride (0.075 g, 2.0 mmol) was added.
The reaction mixture was stirred at room temperature for 5 d
before tri-n-butylamine (0.111 g, 0.6 mmol) was added. A clear
solution resulted. The volatile materials were removed at reduced
pressure, the residue was triturated with hexane to remove any
tri-n-butylamine and the residual material was subjected to flash
chromatography on silica gel using CH2Cl2–MeOH–nBu3N (100 :
10 : 1). The tri-n-butylammonium salt, which contained some free
tri-n-butylamine, was further purified by dissolution in CH2Cl2
and precipitation with hexane, yield 0.105 g (34%) of a white solid.
HRMS (electrospray, TOF ES−]: M-NHBu3 423.0647. Calc. for
C15H16N6O5PS−): 423.0646. 31P NMR (CDCl3, 121 MHz): 55.68;
1H NMR (CDCl3, 300 MHz, MeOH-d4): d 0.93 (9H, t, J 7.3 Hz,
3 × CH3), 1.29–1.41 (6H, m, 3 × CH2), 1.55–1.65 (6H, m, 3 ×
CH2), 2.98–3.04 (6H, m, 3 × CH2), 3.78 (3H, s, N-CH3), 4.12–4.24
(1H, m, H-4ꢀ), 4.25–4.30 (2H, m, OCH2), 4.96 (1H, d, J 5.3 Hz,
(ii) Prepared from the N-benzylamidate 10a. A 1.6 M solution
of n-BuLi in hexane (0.25 mL, 0.39 mmol) was added to a so-
lution of (SP)-2ꢀO-(tert-butyldimethylsilyl)-8-(2-furyl)adenosine-
3ꢀ,5ꢀ-cyclic N-benzylphosphoramidate (10a) (200 mg, 0.33 mmol)
in THF (4 mL) at −78 ◦C. The mixture was stirred for 10 min at
this temperature before carbon disulfide (0.06 ml, 1.0 mmol) was
added and the cooling bath removed. The mixture was stirred for
3 h at room temperature. The volume of the solvent was reduced
to about 1 mL before hexane (40 mL) was added. The precipitate
was collected and dissolved in dry DMF (2 mL), and ammonium
fluoride (75 mg, 2.0 mmol) added. The mixture was stirred under
argon at 40 ◦C for 48 h. The solvent was distilled off and the
crude product purified by flash chromatography on silica gel using
iPrOH–EtOAc–H2O–NH3(aq.) (7 : 7 : 1 : 1); yield: 106 mg (74%)
of a white solid. Spectroscopic data for this compound is given in
the alternative preparation described above.
(Rp)-8-(2-Furyl)adenosine-3ꢀ,5ꢀ-cyclic phosphorothioc acid
tri-n-butylammonium salt (14a)
A 1.6 M solution of n-BuLi in hexane (1.87 mL, 3.0 mmol)
was added to a solution of (SP)-8-(2-furyl)adenosine-2ꢀO-tert-
butyldimethylsilyl)-3ꢀ,5ꢀ-cyclic N-benzylphosphoramidate 10a
(1.650 g, 2.75 mmol) in dry THF (25 mL) at −78 ◦C. The mixture
was stirred under argon at this temperature for 10 min before
CS2 (0.49 mL, 8.25 mmol) was added. The cooling bath was
removed after 10 min. The reaction mixture was stirred at room
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(RP)-8-Adenosine-3ꢀ,5ꢀ-cyclic phosphorothioic acid sodium salt
H-2ꢀ), 5.49 5.57 (1H, m, H-3ꢀ), 5.94 (1H, s, H-1ꢀ), 6.25–6.28 (1H,
m, H-pyr), 6.60–6.62 (1H, m, H-pyr), 6.93–6.95 (1H, m, H-pyr),
8.13 (1H, s, H-2); 13C NMR (MeOH-d4, 75 MHz): d 13.9 (3 ×
CH3), 20.9 (3 × CH2), 26.9 (3 × CH2), 35.7 (N-CH3), 54.0 (3 ×
CH2), 68.5 (d, J 9.8 Hz, OCH2), 72.8 (d, J 7.6 Hz, CH-4ꢀ), 73.1 (d.
J 5.7 Hz, CH-2ꢀ), 77.7 (d, J 6.6 Hz, CH-3ꢀ), 94.2 (CH-1ꢀ), 109.4,
120.1, 121.1, 128.4, 146.3, 151.2, 153.6, 156.8.
(15d)
Compound 15d was prepared from (RP)-adenosine-3ꢀ,5ꢀ-cyclic
phosphorothioic acid tri-n-butylammonium salt (14d) 90% yield.
HRMS (electrospray, TOF ES−): M − Na 344.0211. Calc. for
[C10H11N5O5PS]−: 344.0224. 31P NMR (MeOH-d4, 121 MHz): d
58.39. 31P NMR (D2O, 121 MHz): d 57.02.
(RP)-8-(3-Pyridinyl)adenosine-3ꢀ,5ꢀ-cyclic phosphorothioic acid
tri-n-butylammonium salt (14c)
Diastereomeric synthesis: (RP)- and (SP)-8-(2-furyl)adenosine-
3ꢀ,5ꢀ-cyclic phosphorothioic acid sodium salt (15a) and (15aa)
Compound 14c was prepared as above from (SP)-2ꢀO-
(tert-butyldimethylsilyl)-8-(3-pyridinyl)adenosine-3ꢀ,5ꢀ-cyclic N-
phenylphosphoramidate (11c) in 34%. HRMS (electrospray, TOF
ES−): M-NHBu3 421.0492. Calc. for C15H14N6O5PS]−: 421.0489.
31P (CDCl3, 121 MHz): d 57.17.
8-Furyladenosine22 (16) (0.333g, 1 mmol) was azeotropically dried
in pyridine under reflux. The dried material was dissolved in
dry pyridine (20 mL), and thiophosphoryl chloride (0.169 g,
1 mmol) in dry pyridine (5 mL) was added over 10 min to
the reaction mixture under argon at −8 ◦C. The mixture was
stirred at this temperature for 90 min when a single signal at
58.4 ppm in the 31P NMR spectrum showed full conversion to
the cyclic thiophosphoryl chloride. The reaction mixture was
subsequently added to a vigorously stirred solution of sodium
hydroxide (0.240 g, 6 mmol) in MeCN–H2O (50%, 40 mL) at
room temperature, and the mixture rapidly poured onto crushed
dry ice. When the evolution of CO2 had ceased, the solvents
were removed under reduced pressure with a bath temperature
not exceeding 35 ◦C. The solid precipitate was washed repeatedly
with THF and dissolved in DMF (7 mL) and the solution filtered.
Evaporation of the solvent at reduced pressure furnished the crude
diastereomeric mixture as a solid sodium salt (0.26 g, 60%) in a 1 : 3
ratio of the (RP)- and (SP)-diastereomers. The diastereomers could
be separated by preparative reversed phase chromatography using
H2O–MeOH–HCO2H (80 : 20 : 0.5). 31P NMR (D2O, 81 MHz):
(RP)-isomer 56.20 ppm, (SP)-isomer 55.45 ppm).
(RP)-Adenosine-3ꢀ,5ꢀ-cyclic phosphorothioic acid
tri-n-butylammonium salt (14d)
Compound 14d was prepared as described for 14a from
(SP)-2ꢀO-(tert-butyldimethylsilyl)adenosine-3ꢀ,5ꢀ-cyclic N-benzyl-
phosphoramidate (8) in 41%. HRMS (electrospray, TOF ES−):
344.0231. Calc. for [C10H11N5O5PS]−: 344.0224. 31P NMR
(MeOH-d4, 121 MHz): d 58.16.
(RP)-8-(2-Furyl)adenosine-3ꢀ,5ꢀ-cyclic phosphorothioic acid sodium
salt (15a)
(Rp)–8-(2-Furyl)adenosine-3ꢀ,5ꢀ-cyclic phosphorothioic acid tri-
nbutylammonium salt (14a) (0.600 g, 1.00 mmol) was dissolved in
0.1 M NaOH in MeOH (10 mL). The sodium salt was precipitated
by addition of diethyl ether and collected by filtration, yield 0.389 g
(89%) of a white, solid material. HRMS (electrospray, TOF ES−):
410.0320 Calc. for C14H13N5O6PS]−: 410.0329). 31P (MeOH-d4,
121 MHz): d 58.05; 31P (D2O, 121 MHz): d 56.44; 1H NMR
(MeOH-d4, 200 MHz): 4.25–4.39 (3H, m), 5.15 (1H, d, J 5.2 Hz),
5.45–5.54 (1H, m), 6.24 (1H, s), 6.68–6.71 (1H, m), 7.19–7.21 (1H,
m, ), 7.82–7.83 (1H, m, ), 8.20 (1H, s, H-2); 13C NMR (MeOH-d4,
75 MHz): d 68.5, 72.6, 73.2, 77.6, 94.3, 113.2, 115.3, 120.1, 143.4,
144.4, 146.8, 151.4, 154.0, 156.9.
References
1 K. Taske´n and E. M. Aandahl, Physiol. Rev., 2004, 84, 137–167.
2 S. H. Francis, M. A. Blount, R. Zoraghi and J. D. Corbin, Front. Biosci.,
2005, 10, 2097–2117.
3 W. R. Dostmann, S. S. Taylor, H.-G. Genieser, B. Jastorff, S. O.
Doskeland and D. Ogreid, J. Biol. Chem., 1990, 265, 10484–10491.
4 W. R. Dostmann and S. S. Taylor, Biochemistry, 1991, 30, 8710–8716.
5 B. T. Gjertsen, G. Mellgren, A. Otten, E. Maronde, H.-G. Genieser, B.
Jastorff, O. K. Vintermyr, G. S. McKnight and S. O. Doskeland, J. Biol.
Chem., 1995, 270, 20599–20607.
6 N. Otmakhov and J. E. Lisman, J. Neurophysiol., 2002, 87, 3018–3032.
7 K. Undheim, K. Taske´n, J. Klaveness, G. Langli and V. Bjoernstad, WO
2005123755, 2005; K. Undheim, K. Taske´n, J. Klaveness, G. Langli and
V. Bjoernstad, Chem. Abstr., 2006, 144, 70065.
8 D. Øgreid, R. Ekanger, R. H. Suva, J. P. Miller and S. O. Doskeland,
Eur. J. Biochem., 1989, 181, 19–31.
(RP)-8-(N-Methylpyrrol-2-yl)adenosine-3ꢀ,5ꢀ-cyclic
phosphorothioic acid sodium salt (15b)
Compound 15b was prepared as above from (RP)-8-(N-methyl-
pyrrol-2-yl)-3ꢀ,5ꢀ-cyclic phosphorothioic acid tri-nbutylammo-
nium salt (14b) in 84% yield. HRMS (electrospray, TOF ES−): M −
Na 423.0630. Calc. for [C15H16N6O5PS]−: 423.0646. 31P NMR
(MeOH-d4) d 58.06; (D2O): d 56.42.
9 H. Yokozaki, G. Tortora, S. Pepe, E. Maronde, H.-G. Genieser, B.
Jastorff and Y. S. Cho-Chung, Cancer Res., 1992, 52, 2504–2508.
10 E. M. Aandahl, P. Aukrust, B. S. Ska˚lhegg, F. Mu¨ller, S. Frøland, V.
Hansson and K. Taske´n, FASEB J., 1998, 12, 855–862.
11 K. Taske´n, E. M. Aandahl, P. Aukrust, B. S. Skaalhegg, F. Mueller,
S. Froeland and V. Hansson, WO9848809, 1998; K. Taske´n, E. M.
Aandahl, P. Aukrust, B. S. Skaalhegg, F. Mueller, S. Froeland and V.
Hansson, Chem. Abstr., 1999, 130, 490n.
(RP)-8-(3-Pyridinyl)adenosine-3ꢀ,5ꢀ-cyclic phosphorothioic acid
sodium salt (15c)
12 H.-G. Genieser, W. R. Dostmann, U. Bottin, E. Butt and B. Jastorff,
Tetrahedron Lett., 1988, 29, 2803–2804.
13 F. Eckstein and U. Kutzke, Tetrahedron Lett., 1986, 27, 1657–1660.
14 W. G. Bentrude and J. Thomas, Synthesis, 1984, 27–29.
15 M. Rottla¨nder, L. Boymond, L. Be´rillon, G. Lepretre, G. Varchi, S.
Avolio, H. Laaziri, G. Que`guiner, A. Ricci, G. Cahiez and P. Knochel,
Chem.–Eur. J., 2000, 6, 767–770.
Compound 15c was prepared from (RP)-8-(3-pyridinyl)adenosine-
3ꢀ,5ꢀ-cyclic phosphorothioic acid tri-n-butylammonium salt (14c)
as above in 80% yield. HRMS (electrospray, TOF ES−): M −
Na 421.0495. Calc. for [C15H14N6O5PS]: 421.0489. 31P (MeOH-d4,
121 MHz): d 58.08 ppm.
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View Article Online
16 W. J. Stec, Acc. Chem. Res., 1983, 16, 411–417.
17 J. Beres, W. G. Bentrude, L. Parkanji, A. Kalman and A. E. Sopchik,
J. Org. Chem., 1985, 50, 1271–1278.
18 W. J. Stec, A. Okruszek, K. Lesiak, B. Uznanski and J. Michalski,
J. Org. Chem., 1976, 41, 227–233.
19 D. B. Cooper, C. R. Hall, J. M. Harrison and T. D. Inch, J. Chem. Soc.,
Perkin Trans. 1, 1977, 1969–1980.
20 M. Ikehara and S. Uesugi, Chem. Pharm. Bull., 1969, 17, 348–354;
M. Ikehara, S. Uesugi and M. Kaneko, Nucleic Acid Chem., 1978, 2,
837–841; K. Muneyama, R. J. Bauer, D. A. Shuman, R. K. Robins and
L. N. Simon, Biochemistry, 1971, 10, 2390–2395.
21 L. A. Wozniak and A. Okruszek, Chem. Soc. Rev., 2003, 32, 158–169.
22 F. Eckstein, L. P. Simonson and H.-P. Ba¨r, Biochemistry, 1974, 13,
3806–3810.
Chem. Commun., 1979, 940–941; J. Barniak and W. J. Stec, J. Chem.
Soc., Perkin Trans. 1, 1987, 1645–1656.
24 E. deVroom, G. A. van der Marel and J. H. van Boom, Recl. Trav.
Chim. Pays-Bas, 1987, 106, 577–580.
25 N. Kohyama, T. Katashima and Y. Yamamoto, Synthesis, 2004, 2799–
2804.
26 SMART and SAINT. Area-Detector Control and Integration Software
Version 5.054, Bruker Analytical X-ray Instruments Inc.,Madison,
Wisconsin, USA, 1998.
27 G. M. Sheldrick, SADABS Version 2.01. Program for Empirical
Correction of Area Detector Data, University of Go¨ttingen:, Germany,
2000.
28 G. M. Sheldrick, SHELXTL, Structure Determination Programs
Version 5.10, Bruker Analytical X-ray Instruments Inc., Madison,
Wisconsin, USA, 1997.
23 J. Baraniak, R. W. Kinas, K. Lesiak and W. J. Stec, J. Chem. Soc.,
2080 | Org. Biomol. Chem., 2007, 5, 2070–2080
This journal is
The Royal Society of Chemistry 2007
©