Effective Synthesis of 5-Iodo Derivatives of Pyrimidine Morpholino Nucleosides

Full text of DOI:10.1080/00304948.2018.1462063

Organic Preparations and Procedures International
The New Journal for Organic Synthesis
ISSN: 0030-4948 (Print) 1945-5453 (Online) Journal homepage: http://www.tandfonline.com/loi/uopp20
Effective Synthesis of 5-Iodo Derivatives of
Pyrimidine Morpholino Nucleosides
Ivan P. Vohtancev, Yuliya V. Sherstyuk, Vladimir N. Silnikov & Tatyana V.
Abramova
To cite this article: Ivan P. Vohtancev, Yuliya V. Sherstyuk, Vladimir N. Silnikov & Tatyana
V. Abramova (2018) Effective Synthesis of 5-Iodo Derivatives of Pyrimidine Morpholino
Nucleosides, Organic Preparations and Procedures International, 50:3, 332-342, DOI:
10.1080/00304948.2018.1462063
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Organic Preparations and Procedures International, 50:332–342, 2018
Copyright Ó Taylor & Francis Group, LLC
ISSN: 0030-4948 print / 1945-5453 online
DOI: 10.1080/00304948.2018.1462063
Effective Synthesis of 5-Iodo Derivatives
of Pyrimidine Morpholino Nucleosides
Ivan P. Vohtancev, Yuliya V. Sherstyuk, Vladimir N. Silnikov,
and Tatyana V. Abramova
Institute of Chemical Biology and Fundamental Medicine SB RAS, 8 Lavrentiev
Avenue, Novosibirsk, 630090, Russian Federation
Morpholino derivatives of nucleosides are widely used in medicine and biology as
monomers in the synthesis of DNA or siRNA analogues. They are considered to
be promising agents for anticancer,1 antiviral,2 antibacterial,3 and antiparasitic
therapies4 and for bioanalytical applications.5,6 Morpholino oligonucleotides are
far-reaching candidates in antisense therapy.7 Eteplirsen, morpholino phosphoro-
diamidate oligonucleotide, was approved by FDA for treatment Duchenne
muscular dystrophy in 2016.8 Morpholino nucleoside triphosphates appeared to be
substrates of DNA polymerase b and HIV-1 reverse transcriptase9 and morpholino
thymidine triphosphate was found to be
polymerase.10
a substrate of the Taq DNA
Using the nucleoside derivatives halogenated at the heterocyclic base opens an
easy way to the functionalization of nucleosides, nucleotides, and oligonucleotides.11
The iodine derivatives of nucleosides are most commonly used for further function-
alization in the cross-coupling reaction because they are more reactive than bromine
and chlorine.12
Previously, the synthesis of the N-Tr-protected morpholino nucleoside containing
1a was reported.13 The same authors published a procedure for the preparation of
40-N-Tr- and 20-OCH2-silyl protected morpholino nucleoside containing 5-iodocytosine
as heterocyclic base.14 The method comprised a number of steps including an
introduction and removal of a transient trifluoroacethyl protective group. The iodination
of the heterocyclic base was carried out after the formation of the morpholine ring.
Here, we report the convenient synthesis of the morpholino derivatives of 5-iodouridine
1a and 5-iodocytidine 1b (Figure 1) with a focus on an iodination of uridine and
cytidine at the first step.
In the recently reported procedure,13 the synthesis of 5-iodouridine N-Tr-protected
morpholino nucleoside 1a has been realized by iodination of the N-Tr-protected
Received April 26, 2017; in final form August 30, 2017.
Address correspondence to Yuliya V. Sherstyuk, Institute of Chemical Biology and
Fundamental Medicine SB RAS, Lavrent’ev Ave., 8, Novosibirsk, 630090, Russian Federation.
E-mail: tarasenko@niboch.nsc.ru
Ivan P. Vohtancev and Yuliya V. Sherstyuk contributed equally to this work.
332

Pyrimidine Morpholino Nucleosides
333
O
N
NH2
N
I
I
NH
O
N
O
HO
HO
2`
3`
1`
O
O
4`
6`
N
N
5`
1a
1b
Figure 1 5-Iodopyrimidine N-Tr-morpholino nucleosides.
morpholino uridine in the presence of ICl/K2CO3 in methanol in 83% yield. Later, the
same authors found out that in the case of cytosine containing morpholino monomer, the
iodination protocol described for the uracil containing derivative13 does not work due to
the instability of the trityl group.14 To get 40-N-Tr- and 20-OCH2-silyl protected
morpholino nucleoside containing 5-iodocytosine, they suggested applying the additional
transient trifluoroacetyl protection instead of the trityl group to protect the morpholine
ring nitrogen at the iodination step. The iodination was carried out by iodine/iodic acid
treatment in the mixture of CCl4 and AcOH (1:1) in 91% yield. So, the approach13,14
included a number of additional steps due to the necessity of introduction and removal of
transient trifluoroacetyl protective groups. A silyl protecting group was used for the
50-hydroxy group of the starting ribonucleosides. According to the authors, application of
this group increased the yield of the reaction of the morpholine ring formation from 12%
to 52%.15 A trifluoroacetyl protective group was used to obtain 5-iodocytosine
morpholino nucleoside. The additional synthetic steps also required additional
purifications.
Taking into account the above, we considered that the synthesis of 40-N-Tr-5-
iodopyrimidine morpholino nucleosides 1a,b using 5-iodopyrimidine ribonucleo-
sides as parent compounds for morpholine ring formation would be more straight-
forward. However, no protocol for the synthesis of 5-iodopyrimidine N-Tr-
morpholino nucleosides from the corresponding 5-iodopyrimidine ribonucleosides
has been reported thus far. Recently, we have found conditions that provided mor-
pholino nucleosides in good yields (55–65%) without the transient silyl protec-
tion.16 So, we decided to apply our general protocol for the synthesis of 5-
iodopyrimidine morpholino nucleosides 1a,b (Figure 1) from corresponding 5-
iodopyrimidine ribonucleosides.
First, we prepared 5-iodouridine 2a and 5-iodocytidine 2b from uridine 3a and
cytidine 3b according to the protocol for iodination of 20-deoxycytidine with a slight
modification (Scheme 1).17 In our hands, the yields of 3a and 3b did not exceed
40% and 51%, respectively, after chromatographic purification. The moderate yields
may be explained by the presence of unreacted ribonucleosides 2a,b in the reaction
mixture. If reaction time exceeds 2 hours, some by-product will be formed as regis-
tered by HPLC analysis.

334
Sherstyuk et al.
Scheme 1 Synthesis of 5-iodouridine and 5-iodocytidine by treatment with iodine and iodic acid.
At the next step, we applied the procedure for the morpholino nucleoside synthesis
under careful pH control, providing good yields of target compounds containing
non-halogenated pyrimidine heterocyclic bases.16 Thus, 5-iodinated nucleosides 3a,
Scheme 2 Synthesis of N-tritylated morpholino derivatives of 5-iodouridine and 5-iodocytidine.

Pyrimidine Morpholino Nucleosides
335
b synthesized as depicted in Scheme 1 were used as parent compounds (Scheme 2).
The morpholine ring formation was carried out by a one-pot three-step synthesis
— periodate oxidation, Schiff base formation, and reduction in one flask. To our
surprise, along with the target compounds 4a,b we observed the formation of a
considerable amount of dehalogenated by-products 5a,b during the reduction of the
cyclic intermediate by NaBH3CN under the acidic conditions (pH 3–4) (Scheme 2,
A). The target products 1a,b were obtained after the separation from dehalogenated
by-products by silica gel chromatography after a reduction or tritylation step in
25% and 18% yields, respectively. If the reduction is carried out at pH 5–6, by-
products 5a,b and 6a,b will not form (Scheme 2, B). In this case, tritylated mor-
pholino nucleosides 1a,b were obtained in 55% (1a) and 65% (1b) overall yields,
respectively, starting from 3a,b without any chromatographic purification
(Scheme 2, B).
We tested the stability of compounds 3a,b synthesized according to Scheme 1 in the
presence of NaBH3CN under acidic conditions (pH 3–4). Uridine 2a and cytidine 2b
were formed in 1 hour (Figure 2). Both solutions turned brown because of iodine forma-
tion. Previously, it was shown that the dehalogenation of 5-iodouracil, 5-iodouridine, and
5-iodo-20-deoxyuridine occurs under the action of naphthalene-1,8-diselenol in aq. phos-
phate buffer (pH 7.0) at 37ꢀC.18 Deoxyuridine was obtained by photoirradiation
(302 nm) of 5-iodo-20-deoxyuridine in water in the presence of 2-propanol; NaI signifi-
cantly accelerated the reaction.19 The radical dehalogenation of 5-iodouracil was carried
out in the presence of indium powder,20 tris(trimethylsilyl)silane/2-mercaptoethanol,21
and hypophosphorous acid/NaHCO3.22
Figure 2 HPLC analysis of the interaction between 5-iodocytidine 3b (Scheme 1) and NaBH3CN
under acidic conditions (pH D 3–4) at r.t. HPLC profiles of 5-iodocytidine 3b before addition of
NaBH3CN (A), and reaction between 3b and NaBH3CN under acidic conditions (pH D 3–4) at r.t.
for 1 h (B). Monitoring of a reaction progress was performed on a ProntoSIL 125 C18 column (2 £
75 mm) in a gradient of buffer B (0.1 M TEA–AcOH, pH 7.0, 80% MeCN) in buffer A (0.1 M
TEA–AcOH, pH 7.0, water) with an elution rate of 0.2 mL/min and UV detection at 250, 260, 280,
and 300 nm.

336
Sherstyuk et al.
On the basis of the above facts, we proposed that the unusual instability of the 5-iodopyri-
midine nucleosides in the presence of NaBH3CN under acidic conditions (pH D 3–4) is
promoted by some unidentified inorganic impurities that are present in the parent com-
pounds 3a,b. So, we tried to apply an alternative method of heterocyclic base iodination
for the preparation of 3a,b (Schemes 3, 4) using CAN.21 Following this procedure, 5-
iodouridine 3a was synthesized (Scheme 3). The protocol enables the better purification
of the target compound 3a from inorganic impurities due to silica gel chromatography of
the protected nucleoside 8. Despite the necessity of introduction and removal of the acetyl
protective groups, the overall yield of the compound 3a starting from uridine 2a was still
89%.
Scheme 3 Iodination of uridine in presence of ceric (IV) ammonium nitrate (CAN).
Ph
NH
Ph
NH
Ph
NH
O
O
O
1)(Me)3SiCl, Py
2) BzCl
N
N
I
N
Ac2O, Py
O
3) NH3/H2
O
I2, (NH4)2[Ce(NO3)6]
CH3CN, 80°C
HO
O
N
N
O
O
N
O
O
O
8
O
O
O
OH OH
O
O
O
O
I
O
O
O
O
9
10
12
O
HN
N
O
NH2
N
I2, (NH4)2[Ce(NO3)6]
CH3CN, 80°C
or
NH
O
O
O
O
N
O
N
O
HO
N
O
O
O
O
Ac2O, Py
NaI, (NH4)2[Ce(NO3)6]
CH3COOH, 80°C
O
O
O
O
O
O
O
O
OH OH
2b
8
11
NH2
I
N
NaI, (NH4)2[Ce(NO3)6]
CH3COOH, 80°C
HO
N
O
O
OH OH
3b
Scheme 4 Iodination of cytidine in presence of ceric (IV) ammonium nitrate (CAN).
The halogenated pyrimidine base of 5-iodouridine 3a that was obtained from 20,30,50-tria-
cetyluridine 7 was stable under reducing conditions in the synthesis of the morpholino
nucleoside derivative at pH 3–4. The yield of N-tritylated morpholino 5-iodouridine 1a
was 56% starting from 3a obtained according to Scheme 3.
Since the procedure23 was described only for uracil nucleosides, we have tested sev-
eral methods of obtaining 3b in the presence of CAN from different starting cytidine
derivatives (2b, 10, 11, Scheme 4). We synthesized 20,30,50-triacetyl-N4-benzoylcytidine
10 and N4,20,30,50-tetraacetylcytidine 11 from cytidine 2b.24 Benzoyl protection for the
exocyclic amino group of cytosine was chosen due to its wide application in triphosphate

Pyrimidine Morpholino Nucleosides
337
or oligonucleotide synthesis. Compound 10 was treated with I2 and CAN in acetonitrile at
80ꢀC. In 14 hours, the reaction was completed. After separation of the products of the
reaction mixture we found that compounds 12 and 8 were formed. In the case of iodin-
ation of the fully acetylated cytidine 11 by both I2/CAN in acetonitrile and NaI/CAN in
acetic acid at 80ꢀC, we observed deacetylation and deamination with formation of iodin-
ated uridine 8 as a single product. The iodination of unprotected cytidine 2b in the pres-
ence of NaI/CAN in acetic acid at 80ꢀC was more successful. The yield of 5-iodocytidine
3b after chromatographic purification and precipitation was 55%.
The halogenated pyrimidine base of 5-iodocytidine 3b obtained from cytidine 2b
according to Scheme 4 was stable under reducing conditions in the synthesis of morpho-
lino derivatives of nucleosides at pH 3–4.
In summary, we have synthesized the morpholino derivatives of 5-iodouridine
and 5-iodocytidine from halogenated nucleosides for the first time. The optimization
of the synthesis of 5-iodopyrimidine morpholino nucleosides was based on taking
into account the peculiarities of the behavior of 5-iodo derivatives of pyrimidine
nucleosides obtained by different protocols under reducing conditions. In addition to
the well-known protocol for iodination of uridine in the presence of CAN,23 we
developed a similar procedure for cytidine. Using the parent 5-iodopyrimidines pre-
pared by an alternative method allowed us to achieve the good yields of the target
morpholino nucleosides 1a,b. Based on these results, we can conclude that the
method of iodination of the heterocyclic bases of uridine and cytidine and the purifi-
cation of the iodinated nucleosides contribute significantly into the overall yield of
the target N-tritylated morpholino nucleosides. Our method comprises fewer steps
than the previously published protocol13,14 and is easy to scale up. The resulting
compounds can be used as monomers for oligonucleotide synthesis after the protec-
tion of exocyclic amino group of cytosine, as substrates for DNA polymerases after
triphosphorylation, or for the development of more complex synthetic structures.
Experimental Section
We used NaIO4, (Fluka Chemie, Switzerland), uridine, cytidine (ChemGenes Corpo-
ration, USA), and NaBH3CN (Alfa Aesar, Germany). All other reagents and solvents
were from Sigma–Aldrich (USA) and Reachem (Russia). Thin layer chromatography
(TLC) was carried out on Kieselgel 60 F254 plates (Merck, Germany) in appropriate
solvent systems and visualized by UV-irradiation. Analytical chromatograph Mili-
chrom A-02 (Econova, Novosibirsk) was used for monitoring the progress of reac-
tions; column 2 £ 75 mm, the sorbent ProntoSIL 120–5. The preparative reverse
phase chromatography (RPC) was performed using Porasil C 18 (55–105 mm, 125A)
(Waters, USA). Silica gel was used for column chromatography (35–70 mm, 60A)
(ACROS Organics, USA). Eluent compositions are given in v/v per cent. NMR spec-
tra were recorded on a Bruker AV-300, AV400 and DRX-500 instruments (Bruker,
Germany) in appropriate deuterated solvents at 30ꢀC. Chemical shifts (d) are
reported in ppm relative to appropriate deuterated solvents. Coupling constants J are
reported in Hertz. MALDI–TOF mass spectra were registered on an Autoflex III
mass spectrometer (Bruker Daltonics, Germany) using 2,5-dihydroxybenzoic acid as
a matrix (MALDI–TOF) in positive or negative mode or on an Agilent ESI MSD
XCT Ion Trap (Agilent Technologies, USA) (ESI) in The Core Facility for Mass
Spectrometry at ICBFM SB RAN. The purity of all final compounds (>95%) was
determined by analytical chromatography and by NMR spectra.

338
Sherstyuk et al.
General Procedure for the Synthesis of 5-iodopyrimidine 3a,b According to
Reference 17 (Scheme 1)
The suspension of nucleosides 2a,b (19 mmol) in water (5.7 mL) was treated with HIO3
(9.7 mmol, 1.7 g), AcOH (15.2 mL) and a solution of iodine (11.22 mmol, 2.85 g) in
CCl4 (3.8 mL). The resulting mixture was stirred at 40ꢀC for 2 h until the starting mate-
rial was consumed or some by-product was formed (monitored by HPLC). After that,
water (20 mL) was added. The reaction mixture was cooled to 4ꢀC and filtered. The pre-
cipitate was washed with water (2 £ 10 mL). The combined solutions were diluted with
water (250 ml) and extracted with benzene (3 £ 150 mL). The aqueous layer was evapo-
rated under reduced pressure. The product was purified by RPC in a linear gradient of
EtOH in water (0–30%) to give the product 3a,b.
5-Iodouridine (3a). Yield of 3a from 2a was 40% (7.6 mmol, 2.94 g, yellowish
solid). TLC Rf 0.13 (CH2Cl2: MeOH, 9:1). NMR data were in agreement with those
reported previously.25 1H NMR (500 MHz, DMSO-d6) d 11.70 (s, 1H, NH), 8.48 (s, 1H,
H6), 5.71 (d, 1H, J D 4.3 Hz, H10), 5.45 (d, 1H, J D 4.6 Hz, 20-OH), 5.29 (t, 1H, J D 4.6
Hz, 50-OH), 5.10 (d, 1H, J D 3.6 Hz, 30-OH), 4.00 (dq, 2H, J D 4.4, 27.3 Hz, H30, H20),
3.96 (br s, 1H, H40), 3.62 (dd, 2H, J D 11.5, 57.1 Hz, H50). 13C NMR (125MHz,
DMSO-d6) d 160.61, 150.45, 145.21, 88.33, 84.75, 74.01, 69.42, 68.96, 60.21.
5-Iodocytidine (3b). Yield of 3b from 2b was 51% (9.6 mmol, 3.54g, yellowish
solid). TLC Rf 0.25 (CH2Cl2: MeOH, 4:1). NMR data were in agreement with those
reported previously.25 1H NMR (500 MHz, DMSO-d6) d 8.42 (s, 1H, H6), 7.86 (s, 1H,
NH2), 6.67 (s, 1H, NH2), 5.69 (d, 1H, J D 3.1 Hz, H10), 5.40 (br s, 1H, 20-OH), 5.25 (br s,
1H, 50-OH), 5.02 (br s, 1H, 30-OH), 3.96-3.90 (m, 2H, H30, H20), 3.86-3.81 (m, 1H, H40),
3.62 (dd, 2H, J D 11.9, 74.9 Hz, H50). 13C NMR (125MHz, DMSO-d6) d 163.88, 154.31,
147.88, 89.59, 84.19, 74.54, 69.01, 60.04, 56.86.
Synthesis of N4-benzoylcytydine 9 (Scheme 4).24
Cytidine 2b (20 mmol, 4.86 g) was co-evaporated with pyridine (3 £ 20 mL) to dryness
under reduced pressure. After that, 2b was suspended in dry pyridine (200 mL). After
cooling of the suspension in an ice bath trimethylsilylchloride (100 mmol, 13 mL) was
added dropwise with vigorous stirring. The ice bath was removed and the suspension was
stirred well for 1.5 h at r.t. After cooling of the suspension in an ice bath benzoyl chloride
(40 mmol, 4.7 mL) was added dropwise with stirring. The ice bath was removed and the
suspension was well stirred overnight at r.t. Water (20 mL) was added to the reaction
mixture cooled in the ice bath. After stirring for 30 min, the mixture was treated with
28% aq. NH3 (25 mL). The ice bath was removed and the stirring was continued for 1 h.
The reaction mixture was concentrated under reduced pressure. The residual solvent was
removed by co-evaporation with toluene (3 £ 50 mL). The crude material was suspended
in acetone (300 mL). After filtration, the residual solid was treated with hot water
(300 ml). After cooling, the precipitate was filtered, washed with cold water (2 £ 20 mL)
and dried. The yield of 9 from 2b was 95% (19 mmol, 6.59 g). NMR data were in agree-
ment with those reported previously.26 1H NMR (400 MHz, DMSO-d6) d 11.24 (s, 1H,
NH), 8.51 (d, J D 7.2 Hz, 1H, H6), 8.01 (app d, J D 7.5 Hz, 2H, Bz), 7.61 (app t, J D 7.3
Hz, 1H, Bz), 7.50 (app t, J D 7.6, 2H, Bz), 7.38-7.29 (m, 1H, H5), 5.83 (d, J D 2.8 Hz,
1H, H10), 5.54 (d, J D 4.8 Hz, 1H, 20-OH), 5.20 (t, J D 5.0 Hz, 1H, 50-OH), 5.07 (d, J D
5.7 Hz, 1H, 50-OH), 4.07-3.98 (m, 2H, H30, H20), 3.96-3.91 (m, 1H, H40), 3.81-3.58 (m,
2H, H50). 13C NMR (125 MHz, DMSO-d6) d 167.47, 163.09, 154.75, 145.42, 133.24,
132.80, 128.52, 96.01, 90.32, 84.31, 74.65, 68.72, 59.98.

Pyrimidine Morpholino Nucleosides
339
General Procedure for Acetylation of 2a, 2b, and 9 to Afford 7, 11, and 10 (Scheme 3
and Scheme 4)
Compound 2a, 2b or 9 (10 mmol) was co-evaporated with pyridine (3 £ 10 mL) to dry-
ness under reduced pressure. The dry residue was suspended in pyridine (50 mL). After
cooling of the suspension in an ice bath, acetic anhydride (50 mmol, 4.6 mL for 2a and
9; 60 mmol, 5.7 mL for 2b) was added dropwise with vigorous stirring. The ice bath was
removed and the reaction mixture was well stirred overnight at r.t. After completion of
the reaction, water (20 mL) was added to quench the acetic anhydride.
For the purification of 7 and 10. The reaction mixture was concentrated under
reduced pressure. The crude material was dissolved in CH2Cl2 (300 mL) and washed
with sat. aq. sol. NaHCO3 (300 mL) and water (3 £ 200 mL). The organic layer was
dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was dis-
solved in CH2Cl2 (20 mL) and precipitated with petroleum ether (250 mL).
For the purification of 11. The reaction mixture was concentrated and co-evaporated
with a mixture of water and toluene (3:1; 5 £ 50 mL) and then with toluene (2 £
20 mL). The residue was dissolved in CH2Cl2 (20 mL) and precipitated with mixture of
petroleum and diethyl ether (1:1, 250 mL).
20,30,50-Tri-O-acetyluridine (7). The yield of 7 from 2a was 98% (9.8 mmol, 3.63 g,
white solid). TLC Rf 0.23 (Petr. ether: EtOAc, 1:2). NMR data were in agreement with
those reported previously.27 1H NMR (300 MHz, CD3CN) d 9.52 (s, 1H, NH), 7.47 (d,
J D 8.18 Hz, 1H, H6), 5.90 (d, J D 5.02 Hz, 1H, H10), 5.68 (d, J D 8.09 Hz, 1H, H5),
5.37-5.27 (m, 2H, H20, H30), 4.32-4.18 (m, 3H, H40, H50), 2.04 (s, 3H, CH3), 2.03 (s, 3H,
CH3), 2.01 (s, 3H, CH3). 13C NMR (75 MHz, CD3CN) d 171.33, 170.77, 170.67, 164.14,
151.45, 141.55, 103.58, 88.75, 80.79, 73.53, 70.99, 63.99, 21.00, 20.75, 20.65.
N4-Benzoyl-20,30,50-tri-O-acetylcytidine (10). The yield of 10 from 9 was 97%
(9.7 mmol, 4.59 g, white solid). NMR data were in agreement with those reported previ-
ously.28 1H NMR (400 MHz, DMSO-d6) d 11.37 (s, 1H, NH), 8.19 (d, J D 7.6 Hz, 1H,
H6), 8.00 (app d, J D 7.7 Hz, 2H, Bz), 7.64 (app t, J D 7.5 Hz, 1H, Bz), 7.52 (app t, J D
7.6 Hz, 2H, Bz), 7.40 (d, J D 7.5 Hz, 1H, H5), 5.93 (d, J D 3.7 Hz, 1H, H10), 5.57-5.51 (m,
1H, H20), 5.39 (t, 1H, J D 6.3 Hz, H30), 4.41-4.21 (m, 3H, H40, H50), 2.08 (s, 3H, CH3),
2.07 (s, 3H, CH3), 2.06 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) d 170.18, 169.46,
167.48, 154.30, 146.95, 133.02, 128.54, 96.84, 90.85, 79.21, 72.74, 69.69, 62.89, 20.63,
20.37.
N4,20,30,50-Tetraacetylcytidine (11). The yield of 11 from 2b was 99% (9.9 mmol,
4.07 g, white solid). NMR data were in agreement with those reported previously.29 1
H
NMR (500 MHz, DMSO-d6) d 11.03 (s, 1H, NH), 8.13 (d, J D 7.6 Hz, 1H, H6), 7.26 (d,
J D 7.5 Hz, 1H, H5), 5.91 (d, J D 3.6 Hz, 1H, H10), 5.53-5.49 (m, 1H, H20), 5.39 (t, J D
6.2 Hz, H30), 4.40-4.21 (m, 3H, H40, H50), 2.13 (s, 3H, CH3), 2.09 (s, 3H, CH3), 2.08 (s,
3H, CH3), 2.06 (s, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) d 171.17, 170.12, 169.40,
163.04, 154.27, 146.98, 96.03, 90.84, 79.15, 72.71, 69.69, 62.90, 24.43, 20.57, 20.32.
General Procedure for Iodination of Pyrimidine Nucleosides 2b, 7, 11, 10 (Scheme 3
and Scheme 4).23
Ceric (IV) ammonium nitrate (CAN) was dried over P2O5 under vacuum at 60ꢀC for
4 h.
Method A (I2, CAN). Protected nucleoside 10, 11or 7 (5 mmol) was suspended in
acetonitrile (80 mL); iodine (3 mmol, 0.76 g) and CAN (2.5 mmol, 1.37 g) were added

340
Sherstyuk et al.
to the suspension. The reaction mixture was stirred at 80ꢀC for 1 h (monitored by TLC,
CH2Cl2: acetone, 4:1). After cooling to ambient temperature, the reaction mixture was
evaporated under reduced pressure. The residue was partitioned between a cold mixture
of EtOAc (200 mL), sat. aq. NaCl (100 mL), and 5% NaHSO3 (w/v, 50 mL). The aque-
ous layer was washed with EtOAc (2 £ 100 mL). The combined organic layers were
washed with cold 5% NaHSO3 (w/v, 50 mL), sat. aq. NaCl (150 mL), and water (2 £
150 mL), dried over Na2SO4, filtered and evaporated. The crude product was purified by
silica gel column chromatography using CH2Cl2: acetone (6:1). The yield of 20,30,50-tri-
O-acetyl-5-iodouridine 8 from 7 was 93% (4.65 mmol, 2.31 g). TLC Rf 0.7 (CH2Cl2: ace-
tone, 4:1). NMR data were in agreement with those reported previously.27 1H NMR
(500 MHz, CDCl3) d 9.09 (s, 1H, NH), 7.88 (s, 1H, H6), 6.08-6.05 (m, 1H, H10), 5.34-
5.30 (m, 2H, H20, H30), 4.42-4.31 (m, 3H, H40, H50), 2.23 (s, 3H, CH3), 2.12 (s, 3H, CH3),
2.09 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) d 170.19, 169.73, 169.72, 159.57,
149.99, 143.87, 87.36, 80.49, 73.23, 70.35, 69.70, 63.15, 21.21, 20.61, 20.51.
MethodB(NaI, CAN). Nucleoside 2b or 11 (2 mmol) was suspended in acetic acid
(32 mL); sodium iodide (2.4 mmol, 0.36 g) and CAN (4 mmol, 2.19 g) were added. The
reaction mixture was stirred at 80ꢀC for 30 min (monitored by TLC, CH2Cl2: acetone,
4:1 for 11 and MeOH:CH2Cl2, 1:4 for 2b). After cooling to ambient temperature, the
reaction mixture was evaporated and then co-evaporated with a mixture of EtOH and tol-
uene (2:1; 3 £ 40 mL) and EtOH (3 £ 40 mL) under reduced pressure. The crude product
was purified by silica gel column chromatography using CH2Cl2:MeOH (1:1). 5-Iodocyti-
dine 3b was dissolved in methanol (5 mL) and precipitated with EtOAc (50 mL). The
yield of 3b from 2b was 55% (1.1 mmol, 0.41 g).
Deacetylation of 20,30,50-tri-O-acetyl-5-iodouridine 8 (Scheme 3)
Compound 8 (3.87 mmol, 1.92 g) was dissolved in MeOH (20 ml) and 28% aq. NH3
(20 mL) was added. The reaction was completed in 1 h at ambient temperature
(monitored by TLC, CH2Cl2:MeOH, 9:1). The reaction mixture was evaporated and then
co-evaporated with a mixture of EtOH:EtOAc:toluene (1:1:2; 2 £ 80 mL). The crude
product was purified by silica gel column chromatography using CH2Cl2:MeOH (1:4) to
give the 5-iodouridine 3a in 98% yield (3.83 mmol, 1.42 g), as white solid.
General Procedure for the Synthesis of N-tritylated Morpholino Derivatives
of 5-iodouridine 1a and 5-iodocytidine 1b
The 5-iodonucleosides 3a or 3b (prepared according to23 Scheme 3 and 4, 4 mmol) were
suspended in EtOH (80 mL). A solution of sodium periodate (4.2 mmol, 0.9 g) in water
(4 mL) was added to the nucleoside suspension. After 15 min of stirring, ammonium
biborate tetrahydrate (5.2 mmol, 1.26 g) was added. Triethylamine (TEA) (1.5 mL) was
added until the pH of the resulting mixture was 8.5-9. After 1.5 h, the reaction mixture
was filtered and the precipitate was washed with EtOH (3 £ 1 mL). Sodium cyanoboro-
hydride (5.2 mmol, 0.326 g,) was added to the filtrate. After 1 h the reaction mixture was
adjusted to pH 3–4 with trifluoroacetic acid (TFA). After 2 h, TEA was added to pH 8.0.
The reaction mixture was evaporated and then co-evaporated with a mixture of toluene
and MeCN (1:1; 3 £ 20 mL) under reduced pressure. The resulting oil was dissolved in
dry DMF (16 mL) with addition of TEA (12 mmol, 1.7 mL). Triphenylmethyl chloride
(3.6 mmol, 1 g) was added in portions. The reaction was completed after 1 h (monitored
by HPLC and TLC, CH2Cl2:MeOH, 9.5:0.5 and iPrOH:H2O, 4:1). After addition of

Pyrimidine Morpholino Nucleosides
341
EtOH (5 mL), the reaction mixture was concentrated to half volume and then was poured
into water (300 mL) with vigorous stirring. The precipitate formed was separated by fil-
tration. After drying, the crude product was dissolved in CH2Cl2 (10 mL) and precipitated
with petroleum ether (150 mL). The yield of 1a was 55% (2.2 mmol, 1.31 g, white solid).
The yield of 1b was 65% (2.6 mmol, 1.54 g, white solid).
In the case of 3a or 3b prepared according to Scheme 1. The reaction mixture was
adjusted to pH 3–4 with TFA. After 2 h, TEA was added to pH 8.0. The reaction mixture
was evaporated and co-evaporated with a mixture of toluene and MeCN (1:1; 3 £
20 mL) under reduced pressure. The resulting oil was dissolved in dry DMF (16 mL)
with addition of TEA (12 mmol, 1.7 mL). Triphenylmethyl chloride (3.6 mmol, 1 g) was
added in portions. The reaction was completed after 1 h (monitored by HPLC and TLC,
CH2Cl2:MeOH, 9.5:0.5 and iPrOH:H2O, 4:1). After addition of EtOH (5 mL), the reac-
tion mixture was concentrated to half volume and then was poured into water (300 mL)
with vigorous stirring. The precipitate was separated by filtration. After drying, the crude
product was dissolved in CH2Cl2 and precipitated with petroleum ether. The resulting
mixture of 1a and 6a was separated by silica gel column chromatography in a gradient of
acetone in CH2Cl2 (0!25%). The yield of 1a from 3a was 25% (1 mmol, 0.59 g). The
yield of 1b from 3b was 18% (0.7 mmol, 0.43 g).
N-Tritylated morpholino derivative of 5-iodouridine 1a. TLC Rf 0.57 (CH2Cl2:
MeOH, 9.5:0.5). NMR data were in agreement with those reported previously.13 1
H
NMR (500 MHz, DMSO-d6) d 11.70 (s, 1H, NH), 7.86 (s, 1H, H6), 7.15-7.60 (m, 15H,
Tr), 5.94 (dd, J D 2.0, 9.4 Hz, 1H, H60), 4.75 (t, J D 6.0 Hz, 1H, 20-CH2OH), 4.15 (m,
1H, H20), 3.40 (t, J D 5.3 Hz, 2H, 20-CH2OH), 3.20 (app d, J D 11.4 Hz, 1H, H50), 2.98
(app d, J D 11.8 Hz, 1H, H30), 1.42 (app t, J D 11.2 Hz, 1H, H30), 1.32 (app t, J D
10.5 Hz, 1H, H50). 13C NMR (125 MHz, DMSO-d6) d 160.45, 149.51, 144.31, 141.12,
129.28-126.20, 80.52, 76.98, 76.27, 69.57, 61.93, 51.70, 48.95. MS (MALDI-TOF) m/z:
[M-H]¡ calcd. for E28H25IN3O4 594.089, found 594.035.
N-Tritylated morpholino derivative of 5-iodocytidine 1b. An analytical sample was
1
re-crystalized from CH2Cl2-hexane. TLC Rf 0.51 (CH2Cl2: MeOH, 9.5:0.5). H NMR
(300 MHz, CDCl3) d (ppm): 8.41 (br s, 1H, NH2), 7.55 (s, 1H, H6), 7.52-7.03 (m, 15H,
Tr), 6.11 (d, J D 8.17 Hz, 1H, H60), 5.76 (br s, 1H, 20-CH2OH), 4.36-4.22 (m, 1H, H20),
3.67-3.49 (m, 2H, 20-CH2OH), 3.44 (app d, J D 10.08 Hz, 1H, H50), 3.17-2.99 (m, 1H,
H30), 1.48-1.16 (m. 2H, H50, H30). 13C NMR (75 MHz, CDCl3) d 163.57, 154.26, 146.71,
145.14, 129.33, 128.01, 126.58, 81.97, 78.06, 76.87, 63.68, 56.99, 52.91, 49.06. MS
(ESI) m/z: [M-H]¡ calcd. for E28H26IN4O3 593.105, found 593.092.
Anal. Calcd for C28H27IN4O3: C 56.57, H 4.58, N 9.43. Found: C 56.72, H 4.51, N 9.43.
Acknowledgments
The work was supported by the Russian Foundation for Basic Research (grant No.16-34-00718
to Yu.V. Sherstyuk) and by the Russian State funded budget project 57 (No. 0309-2016-0001).
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