Preparation of potential cell-permeant nucleoside-2′,3′-cyclic phosphate precursors

Article abstract of DOI:10.1080/15257770701257236

Uridine-3′-phosphorothiolate triesters bearing lipophilic moieties were prepared via Michaelis-Arbuzov chemistry. Subsequent deprotection of the S-cholesteryl phosphorothiolate triester afforded the corresponding diester which underwent spontaneous Cycliz

Full text of DOI:10.1080/15257770701257236

Nucleosides, Nucleotides, and Nucleic Acids, 26:245–254, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701257236
PREPARATION OF POTENTIAL CELL-PERMEANT
NUCLEOSIDE-2ꢀ,3ꢀ-CYCLIC PHOSPHATE PRECURSORS
Simone Battaggia, Emma E. Smith, and Joseph S. Vyle
School of
2
Chemistry and Chemical Engineering, Queen’s University Belfast, Belfast, United Kingdom
Uridine-3 ꢁ-phosphorothiolate triesters bearing lipophilic moieties were prepared via Michaelis-
2
Arbuzov chemistry. Subsequent deprotection of the S-cholesteryl phosphorothiolate triester afforded
the corresponding diester which underwent spontaneous Cyclization to cleanly afford uridine 2 ꢁ,3 ꢁ-
cyclic phosphate. This transesterification reaction could be expedited by treatment with iodine under
mild, neutral conditions.
Keywords Michaelis-Arbuzov; cell-permeant; phosphorothiolate; cyclic phosphate;
precursor
INTRODUCTION
Recent interest in 2ꢁ,3ꢁ-cyclic nucleotide 3ꢁ-phosphodiesterase (EC
3.1.4.37, CNP) results from its intracellular templating role in micro-
tubule formation[1] and its maintenance of extracellular myelination by
oligodendrocytes.[2] CNP represents 4% of total myelin proteins and its
malfunction in oligodendrocytes is a key feature of several disease states
including Multiple Sclerosis, lead poisoning and schizophrenia.[3,4]
The two splice isoforms—CNPI (46 kD) and CNPII (48 kD)—are
highly modified posttranslationally and are heterogenous in size and
charge.[5] Such diversity results in differential subcellular localisation[6] and
also can interfere with immunohistochemical staining of the appropriate
epitopes.[3,7] Although CNP mediated hydrolysis of 2ꢁ,3ꢁ-cyclic nucleotides
into the corresponding 2ꢁ-monoesters has been investigated chemically,[8]
the in vivo relevance of this activity is still uncertain as such substrates have
not been detected in the brain.
Received 25 July 2006; accepted 20 November 2006.
This work was funded by the MRC (grant reference G0100461), the School of Chemistry, QUB
and The Royal Society (grant reference 574006). We acknowledge Marie Migaud (QUB, Chemistry and
Chemical Engineering) for useful discussions.
Simone Battaggia current address: Bristol-Myers Squibb, Watery Lane, Swords, Co.Dublin, Ireland.
Address correspondence to Jospeh S. Vyle, School of Chemistry and Chemical Engineering,
Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, UK. E-mail:
j.vyle@qub.ac.uk
245

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S. Battaggia et al.
The development of membrane-permeant nucleotide substrate ana-
logues of CNP would enable more definitive intracellular spatial visualisa-
tion of CNP, and could potentially form the basis of therapeutic intervention
in diseases involving CNP malfunction. However, intracellular delivery of
such analogues has not, to the authorsꢁ knowledge, been reported. This
can be attributed to the polar nature of the anion and also the reactivity
of five-membered cyclic phosphates.[9]
The simplest and most well-established strategy for delivering anionic
nucleotides across cell membranes is to append lipophilic moieties to
nonessential functionalities of bioactive compounds such as nucleoside 3ꢁ,5ꢁ-
cyclic phosphate second messengers, for example, N 6,O 2ꢁ-dibutyryl guano-
sine 3ꢁ,5ꢁ-cyclic phosphate.[10] More elegant and effective prodrug strate-
gies for delivering charge-neutralized mononucleotides use “programmable
linkers,” which only release the drug upon internalization by target cells and
exposure to cytoplasmic esterases or low endosomal pH.[11]
Phosphorothiolates represent one potential programmable linker as
the P-S linkage is sensitive to the endosomal pH. In addition, enhanced
intramolecular trans-esterification rates of the phosphorothiolate-linked
dinucleoside UpsU compared with its natural congener UpU have been
documented previously.[12] We report here our results concerning the
preparation of lipophilic 3ꢁ-O-phosphorothiolate derivatives of uridine and
subsequent transformation of the 3ꢁ-O,S-cholesteryl diester to the corre-
sponding 2ꢁ,3ꢁ-cyclic phosphate under mild neutral conditions.
RESULTS AND DISCUSSION
First reported in 1974 by Hata and Sekine,[13] the synthesis of nucleoside
S-phenyl phosphorothiolate esters via directed Michaelis-Arbuzov (M-A)
chemistry has yielded intermediates in the preparation of nucleoside phos-
phoramidates, pyrophosphates, and phosphate diesters.[14] Subsequent
development of this chemistry has principally focused on the permanent
installation of inter- and intranucleoside phosphorothiolate diesters.[15]
Activated disulfides bearing lipophilic moieties (1a, b; Figure 1) were
prepared for this study.
Disulfide 1a was prepared according to the procedure of Eckstein
and coworkers.[16] The previously unreported 1b was synthesized by DCC-
mediated coupling of 11-bromoundecanoic acid with 1-dodecylamine and
transformation of the resultant 11-bromoundecamide into the correspond-
ing thiol via the thiouronium salt.[17] High yields from this scheme were
critically dependent on exclusion of oxygen during final alkaline hydrolysis
to liberate the thiol and during its subsequent isolation and transformation
to 1b.
We previously have described the utility of o-methylbenzyl phosphite
intermediates for the synthesis of deoxyribonucleoside phosphorothiolate

Cell-Permeant Nucleoside-2ꢁ,3ꢁ-Cyclic Phosphate Precursors
247
FIGURE 1 Activated disulfides used in Michaelis-Arbuzov reactions.
triesters.[18] The acid sensitivity of such intermediates precluded the
application of this strategy for the preparation of lipophilic appended
ribonucleoside-phosphorothiolates. We, thus, sought to apply a strategy
starting from the activated H-phosphonate (2; Scheme 1).[19] The sensitivity
of 5ꢁ-S-ribonucleoside phosphorothiolates towards cleavage in the presence
of fluoride or base[12,20] led us to utilize the acid-sensitive 2ꢁ-O-Fpmp-
protecting group for the chemistry.
Following activation of the H-phosphonate
2
with N ,O-bis
(trimethylsilyl)acetamide, the trimethylsilylphosphite triester derivative of
uridine was successfully treated in situ under ambient conditions with 1a or
1b to afford the corresponding S-alkyl uridine-3ꢁ-phosphorothioate triesters
3a (96% pure by 31P NMR) and 3b (94%) after 24 hours or 12 hours,
respectively. The only side-product observed was the phosphorothiolate
SCHEME 1 Preparation of phosphorothiolate-derivatized nucleoside. Reagents and conditions: i) N ,O-
Bistrimethylsilyl acetamide (10 eq), CDCl3, 25◦C, 2.5 hours; ii) 1a (20 hours) or 1b (12 hours) (1.5 eq),
CDCl3, 25◦C; iii) TCA (5 eq), pyrrole (20 eq), DCM, 25◦C, 1 minute; v) sat. NH3 in MeOH, 25◦C, 26
hours; vi) HCl (aq), pH 3, MeOH/CHCl3 (4.4:1), 25◦C, 2 hours.

248
S. Battaggia et al.
resulting from removal of the S-alkyl function. 3a was subsequently isolated
in 85% yield following chromatography. In contrast, purification of 3b
resulted in highly attenuated yields (typically 40%) principally attributable
to its low solubility.
The 2ꢁ-acetal moiety retained its integrity during acid-mediated detrity-
lation of 3a and 3b under anhydrous conditions and, thus, afforded the
corresponding 5ꢁ-hydroxy nucleotides in 84% and 90% yields by 31P NMR.
However spontaneous intramolecular cyclization of the phosphorothiolate
triesters to the corresponding 3ꢁ,5ꢁ-cyclic phosphate (t1/2 15 hours) was
observed and only moderate yields (45% from 3a; 22% from 3b) were
recovered following purification by silica gel column chromatography.
Immediate removal of the 2-cyanoethyl group under mild conditions to
yield the corresponding phosphorothiolate diesters, thus, was performed in
order to inhibit the transesterification reaction. This proceeded cleanly for
both products by 31P NMR, although the N -dodecyl-S-undecanamide diester
(from 3b) proved intractable. The 2ꢁ-protected S-cholesteryl diester (from
3a) was isolated in 88% yield and the phosphorothiolate linkage was robust
during work-up and handling. The 2ꢁ-ketal was hydrolysed cleanly at pH 3 to
give 4 in 62% yield and 100% purity. The fully deprotected nucleotide was
stored frozen at neutral pH (6.8). This significantly reduced (although did
not eliminate) transesterification to the 2ꢁ,3ꢁ-cyclic phosphate.
Cyclization was further expedited using iodine at neutral pH
(Scheme 2);[20] competing hydrolysis to the phosphate monoester is not
observed due to the high effective molarity (ca. 107 M) of the vicinal
hydroxyl.[21] Exclusive formation of uridine 2ꢁ,3ꢁ-cyclic phosphate in 95%
yield by 31P NMR after 2 hours was observed (Figure 2).
We currently are seeking to employ lipophilic nucleoside and nu-
cleotide phosphorothiolates, in particular cholesteryl derivatives,[22] as
cyclic phosphate precursors for the investigation of the role of CNP in
demyelination and also to the enigmatic function of biochemically related
enzymes/domains in yeast and virus.[22]
SCHEME 2 Cyclization of S-cholesteryl phosphorothiolate. Reagents and conditions: i) I2 (1eq),
aqueous 0.1M TEAA (aq) /CD3OD/CDCl3 (34:38:28 v/v/v), pH 6.8, 25◦C, 2 hours.

Cell-Permeant Nucleoside-2ꢁ,3ꢁ-Cyclic Phosphate Precursors
249
FIGURE 2 31P NMRs showing cyclization of 4 before (a) and after (b) addition of I2.
EXPERIMENTAL
General Methods
NMRs were recorded on General Electric QE 300, Brucker DPX 300
or Brucker DRX 500 spectrometers. Electrospray (MS-ES+) mass spectro-
scopies were performed on a VG Auto Spec Mass Spectrometer operating at
8 kV and using 3-nitrobenzylalcohol as the matrix.
Columns were packed with dry silica (Merk 7736 Kieselgel 60 H). TLCs
were carried out on Merk Kieselgel 60254 and the spots visualized at 254 nm.
Chloroform and DCM were refluxed and distilled from phosphorus pen-
toxide (Aldrich, UK) and passed through activated basic alumina (Aldrich,
UK) before use. CDCl3 (Aldrich, UK) was passed through activated basic
alumina before use. EtOH and MeOH (GPR, Fisher, UK) were refluxed with
˚
the corresponding magnesium alkoxide and fractionally distilled onto 3 A
molecular sieves (Aldrich, UK). All other reagents were used as supplied
from Fluka/RdH (UK).
N-dodecyl-11-(5-nitropyridyldisulfanyl)undecanamide (1b). To a vigor-
ously stirred suspension of 11-bromoundecanoic acid (500 mg, 1.88 mmol)
and N -hydroxysuccinimide (230 mg, 2.06 mmol) in DCM (10 ml) was added
a solution of N,N ꢁ-dicyclohexylcarbodiimide (770 mg, 3.77 mmol) in DCM
(5 ml) under anhydrous conditions at 25◦C. A clear solution was obtained
after 5 minutes and stirring was maintained for an additional 2 hours during
which precipitation of N ,N ꢁ-dicyclohexylurea was observed. The reaction
mixture was then filtered under an inert atmosphere and the filtrate added
to a stirred solution of dodecylamine (690 mg, 3.76 mmol) and DMAP
(10 mg, 0.09 mmol) in anhydrous DCM (10 ml). The reaction mixture was

250
S. Battaggia et al.
heated at 35◦C for 2 hours. Dichloromethane (40 ml) was added and the
diluted solution successively washed with water (3 × 150 ml) and brine
(60 ml), dried over sodium sulfate and filtered. Removal of the solvent
under vacuum afforded a white solid residue which was further purified
by triturating with hexane (200 ml). 11-Bromo-(N -dodecyl)undecanamide
was isolated as a white solid (772 mg, 95%). A solution of the amide
(25 mg, 0.58 mmol) and thiourea (48 mg, 0.63 mmol) in ethanol (15 ml)
was refluxed for 6 hours in the absence of oxygen. The reaction mixture
was then allowed to cool to 20◦C and a 0.2 M solution of sodium hydroxide
in argon-saturated ethanol (3 ml) added. The solution was heated under
reflux for 2 hours, cooled to room temperature and acidified to pH 3 by
addition of dilute HCl (aq). The solution was reduced in vacuo to give a
solid residue, which was dissolved in DCM (70 ml) and washed with water
(6 × 70 ml) followed by brine (100 ml). The organic phase was then dried
over sodium sulfate, filtered, and concentrated under reduced pressure to
yield the 11-mercapto-(N -dodecyl)undecanamide as a white solid (205 mg,
92% yield). The mercaptan (820 mg, 2.13 mmol) was dissolved in argon
saturated DCM/methanol (40 ml, 85:15 v/v). A solution of bis(2-thio-5-
nitropyridine)disulfide (1 g, 3.22 mmol) in argon-saturated DCM/methanol
(60 ml, 50:50 v/v), was added dropwise over 10 minutes. Acetic acid
(2.5 ml) was then added and the resulting orange solution stirred at 45◦C
for 48 hours. The solvent was removed in vacuo, the residue dissolved in
chloroform (150 ml) and successively washed with 2 M aqueous sodium
carbonate (20 ml), water (2 × 250 ml) and brine (40 ml). The organic phase
was dried over sodium sulfate, filtered, and concentrated under vacuum.
The solid obtained was further purified by triturating with acetonitrile
(15 ml × 3), recovering each time the insoluble portion after centrifugation
(15,000 rpm, 30 minutes). The desired product was isolated as a white
powder after removal of the solvent in vacuo (795 mg, 64% yield).
1H NMR (300 MHz, CDCl3) δH: 0.78–0.82 (3H, m, CH3(CH2)11),
1.67–1.85 (36H, m, (CH2)18), 2.07 (2H, t, J = 7.5 Hz, CH2C(O)NH), 2.76
(2H, t, J = 7.3 Hz, CH2SS), 3.16 (2 H, dt, J1 = 6.7 Hz, J2 = 6.4 Hz,
CH2NHCO), 5.30 (1H, broad s, C(O)NH), 7.85 (1 H, d, J = 8.8 Hz, H3
Py-NO2), 8.33 (1 H, dd, J1 = 8.8 Hz, J2 = 2.5 Hz, H4 Py-NO2), 9.20 (1H,
d, J = 2.5 Hz, H6 Py-NO2). 13C NMR (75 MHz, CDCl3) δC: 13.10, 21.67,
24.77, 25.91, 27.42, 27.95, 28.06 (2C), 28.87 (2C), 28.32 (2C), 28.54 (2C),
28.62 (2 C), 28.68 (2 C), 30.89, 35.90, 38.03, 38.49, 76.19, 103.00, 118.09,
130.53, 144.08, 179.50, 171.94. Calc. MS-ES+, 5.60×108eV: 541 [M+H], 563
[M+Na].
S-Thiocholesteryl-2-cyanoethyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-(1-fluoro-
phenyl-4ꢁ-methoxypiperidyl-4ꢁ-yl)uridine 3ꢁ-phosphorothiolate (3a). To a
solution of 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2-cyanoethyl-2ꢁ-O-(1-fluorophenyl-
4ꢁ-methoxypiperidyl-4ꢁ-yl) uridine 3ꢁ-(hydrogen H-phosphonate)[20] (2;
136 mg, 0.16 mmol) in CDCl3 (1.2 ml), was added N ,O-bis(trimethylsilyl)

Cell-Permeant Nucleoside-2ꢁ,3ꢁ-Cyclic Phosphate Precursors
251
acetamide (0.39 ml, 1.6 mmol) at 25◦C. The reaction was maintained under
these conditions for 1 hour after which time complete reaction to the
putative phosphite triester intermediate was inferred by 31P NMR analysis
(δ 130.0 and 129.2 ppm). Solid disulfide 1a (130 mg, 0.24 mmol) was added
in situ with vigorous mixing and the reaction mixture maintained under
ambient conditions for 20 hours. The reaction was subsequently diluted
with ethyl acetate (40 ml) and washed with a 2 M NaHCO3 (aq) (2 × 70
ml), water (2 × 70 ml) and brine (20 ml). The organic phase was dried over
sodium sulfate, filtered, and reduced in vacuo. The residue was purified by
silica gel chromatography eluting with a gradient of methanol (0–3% v/v)
in DCM. 3a was isolated as a cream foam (173 mg, 85% yield).
1
31P NMR (121 MHz, CDCl3) δP: 29.95 and 29.38. H NMR (300 MHz,
CDCl3) δH: 0.55–2.07 (43H, m, aliphatic chol.), 3.09–3.11 (3H, 2s,
OCH3-Fpmp), 2.33–3.18 (11H, m, PSCH, (CH2CH2)2N + OCH2CH2CN),
3.31–3.49 (2H, m, H5ꢁ + H5ꢁꢁ), 3.70 (6H, s, OCH3-DMTr), 4.30–4.11 (2H, m,
OCH2CH2CN), 4.35–4.39 (1H, m, H4ꢁ), 4.91–4.82 (1H, m, H3ꢁ), 4.96–5.08
(1H, m, H2ꢁ), 5.14–5.16 (1H, m, H5), 5.16–5.22 (1H, m, vinylic chol.),
6.10–6.22 (1H, m, H1ꢁ), 6.74–7.24 (17H, m, ArH-DMTr + ArH-Fpmp), 8.60
(1H, broad s, N3H), 8.66–8.70 (1H, m, H6).MS-ES+, 9.31×106 eV: 1273
[M+H] 1295 [M+Na].
S-(N-dodecyl-11-undecanamidyl)-2-cyanoethyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)
-2ꢁ-O-(1-fluorophenyl-4ꢁ-methoxypiperidyl-4ꢁ-yl)uridine 3ꢁ-phosphorothiolate
(3b). Prepared as above except that the M-A reaction was complete within
12 hours (86 mg, 44% yield).
Rf (dichloromethane/methanol 9:1) = 0.56. 31P NMR (121 MHz,
1
CDCl3) δp: 31.05 and 30.29. H NMR (300 MHz, CDCl3) δH: 0.87 (3H,
t, J = 6.4 Hz, (CH2)11CH3), 1.25–2.10 (36H, m, (CH2)8CH2C(O)NH +
(CH2)10CH2NHC(O)), 2.14 (2H, t, J = 7.6 Hz, CH2C(O)NH), 2.75 (2H,
t, J = 6.3 Hz, OCH2CH2CN), 2.62–3.18 (10H, m, (CH2CH2)2N + PSCH2),
3.05–3.28 (5H, m, C(O)NHCH2 + OCH3-Fpmp), 3.40–3.65 (2H, m, H5ꢁ +
H5ꢁꢁ), 3.80 (6H, s, OCH3-DMTr), 4.18–4.36 (2H, m, OCH2CH2CN),
4.31–4.44 (1H, m, H4ꢁ), 4.90–5.01 (1H, m, H3ꢁ), 5.09–5.18 (1H, m, H2ꢁ),
5.27–5.30 (1H, m, H5), 5.42–5.51 (1H, broad s, C(O)NH), 6.27–6.30 (1H,
m, H1ꢁ), 6.84–7.34 (17H m, ArH-DMTr + ArH-Fpmp), 7.65–7.80 (1H, m,
H6), 8.98–9.04 (1H, 2 broad s, N3H). MS-ES+, 2.20 × 106eV: 1256 [M+H],
952 [M–DMTr+H].
Ammonium salt of S-Cholesteryl-uridine-3ꢁ-O-phosphorothiolate (4). To
a stirred solution of 3a (163 mg, 0.2 mmol) in DCM (2 ml) was added a
1 M solution of trichloroacetic acid in anhydrous DCM (1 ml) under an
inert atmosphere at room temperature. The resulting deep orange solution
was maintained under these conditions for 3 minutess and pyrrole (185 µl,
4 mmol) added. After 20 minutes, the reaction mixture had decolorized
and was immediately diluted with ethyl acetate (150 ml), washed with 2
M NaHCO3 (aq) (2 × 300 ml), water (3 × 200 ml) and brine (40 ml).

252
S. Battaggia et al.
The organics were then separated, dried over sodium sulfate, filtered
under reduce pressure, and concentrated in vacuo. The crude detritylated
intermediate was purified by silica gel column chromatography eluting
with 0–3% methanol in DCM to yield a white solid (87 mg, 45%) and
used immediately in the subsequent step. To a solution of this detritylated
nucleotide (61 mg, 0.06 mmol) was added methanolic ammonia (saturated
at −15◦C) in methanol (1 ml), and the reaction mixture maintained at am-
bient temperature for 28 hours. Evaporation of the volatiles under reduced
pressure afforded the desired 2ꢁ-Fpmp-protected phosphorothiolate diester
(51 mg, 88%). The phosphorothiolate diester derivative (30 mg, 0.03 mmol)
was dissolved in 4.4:1 (v/v) methanol:CHCl3 (5.4 ml) HCl (aq) added until
the pH reached 3 (ca. 1 ml) and stirred under ambient conditions for 6
hours. Solvent was removed in vacuo and following coevaporation with
acetonitrile. Hydrolysed Fpmp ketal was removed by trituration with
diethyl ether (8 ml) to yield the product (4) (15 mg, 62% yield) which
was stored frozen in 100 mM triethylammonium acetate (pH 6.8). 31P
1
NMR (121 MHz, CDCl3/CD3OD 6:4) δP : 25.24; H NMR (300 MHz,
CDCl3/CD3OD 6:4) δH: 0.64–2.47 (43H, m, aliphatic chol.), 2.96–3.07 (1H,
m, PSCH), 3.75–3.86 (2H, m, H5ꢁ + H5ꢁꢁ), 4.26–4.37 (2H, m, H3ꢁ + H4ꢁ),
4.70–4.75 (1H, m, H2ꢁ), 5.29–5.32. (1H, m, vinylic chol.), 5.69 (1H, d, J =
8.1 Hz, H5), 5.91 (1H, d, J = 5.3 Hz, H1ꢁ), 7.92 (1H, d, J = 8.1 Hz, H6).
MS-ES-, 9.31×10 6eV: 914 [M-].
Triethylammonium salt of uridine-2ꢁO,3ꢁO-cyclic phosphate (5). A solu-
tion of the triethylammonium salt of phosphorothiolate monoester deriva-
tive 4 (15 mg, 0.02 mmol) in water, buffered at pH 6.8 in triethylammonium
acetate 0.1 M, deuterated methanol and deuterated chloroform (1 ml,
34:38:28 v/v/), was treated with elemental iodine (5 mg, 0.02 mmol) at
25◦C. The reaction was monitored by TLC (dichloromethane and methanol
(6:4 v/v + 0.2% of triethylamine)). After 2 hours the reaction was 95%
complete and the product characterized by co-injection on C18 RP-HPLC
with an authentic sample purchased from Aldrich.
31P NMR (202 MHz, 0.1M TEAA, methanol and dichloromethane
34:38:28:4) δp: 22.21.
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