Versatile synthesis of 2′-amino-2′-deoxyuridine derivatives with a 2′-amino group carrying linkers possessing a reactive terminal functionality

Article abstract of DOI:10.1016/j.tet.2016.07.061

2,2′-Anhydrouridine has been successfully converted into the appropriate 2′-amino-2′-deoxyuridine derivatives in a reaction with isothiocyanates obtained from amino acids or α,ω-diaminoalkanes. The initially formed oxazolidine-2-thione ring is cleaved under basic conditions into the corresponding 2′-amino(substituted)-2′-deoxyuridine derivatives. The implemented additional terminal functionality in the substituent attached to the 2′-amino group allows further modifications with e.g., fluorophore moiety.

Full text of DOI:10.1016/j.tet.2016.07.061

Accepted Manuscript
Versatile synthesis of 2′-amino-2′-deoxyuridine derivatives with a 2′-amino group
carrying linkers possessing a reactive terminal functionality
Andrzej Gondela, Mateusz D. Tomczyk, Łukasz Przypis, Krzysztof Z. Walczak
PII:
S0040-4020(16)30719-0
10.1016/j.tet.2016.07.061
TET 27957
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 20 February 2016
Revised Date: 8 July 2016
Accepted Date: 25 July 2016
Please cite this article as: Gondela A, Tomczyk MD, Przypis E, Walczak KZ, Versatile synthesis of 2′-
amino-2′-deoxyuridine derivatives with a 2′-amino group carrying linkers possessing a reactive terminal
functionality, Tetrahedron (2016), doi: 10.1016/j.tet.2016.07.061.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Versatile synthesis of 2’-amino-2’-deoxyuridine derivatives with a 2’-amino group
carrying linkers possessing a reactive terminal functionality
Andrzej Gondelaa,b, Mateusz D. Tomczyka, Łukasz Przypisa, Krzysztof Z. Walczaka*
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian
University of Technology, B. Krzywoustego 4, 44-100 Gliwice, Poland
Keywords: 2,2’-anhydrouridine, 2’-amino-2’-deoxyuridine, isothiocyanates, ring
opening, fluorophores
Abstract: 2,2’-Anhydrouridine has been successfully converted into the appropriate 2’-
amino-2’-deoxyuridine derivatives in a reaction with isothiocyanates obtained from amino
acids or α,ω-diaminoalkanes. The initially formed oxazolidine-2-thione ring is cleaved under
basic conditions into the corresponding 2’-amino(substituted)-2’-deoxyuridine derivatives.
The implemented additional terminal functionality in the substituent attached to the 2’-amino
group allows further modifications with e.g. fluorophore moiety.
1. Introduction
Since the discovery of puromycin, an amino nucleoside that inhibits protein synthesis, the
activity of nucleosides possessing an amino group in the sugar moiety is well documented and
several methods of their synthesis have been developed. The activity of 2’-amino-2’-
deoxynucleosides against bacteria, viruses and mycoplasma was ascertained in both the
purine and pyrimidine series.1-6 The analogues of 2’-amino-2’-deoxyuridine (2AU)
containing a 3’- or 2’-hydroxyamino group are cytotoxic to murine leukaemia L1210 cells.7
Novel ribozymes containing 2AU instead of uridine were more active in in vitro ligation of
oligodeoxynucleotides.8-11
b Present address: Selvita S.A., Bobrzyńskiego 14, 30-348 Kraków, Poland. E-mail: agondela@gmail.com *Corresponding author: Krzysztof
Z. Walczak, Tel.:+4832 2371308; Fax: +4832 2372094. E-mail: Krzysztof.Walczak@polsl.pl

ACCEPTED MANUSCRIPT
The analogues of 2AU 5’-diphosphate were considered to be agonists of the human P2Y6
receptor, a member of the G-protein coupled receptors.12-14 Another practical application of
amino nucleosides related to the presence of an amino group, is their usage as a conjugation
site for the construction of linkers in oligonucleotides chemistry.15,16 Using this strategy the 2-
20
21
furanyl moiety,17,18 amino acids,19 porphyrins and coumarin were incorporated into the
sugar ring of 2AU and applied as components of antisense oligonucleotides able to improve
drug efficacy by enhancement of resistance to chemical and enzymatic degradation.22,23 Due
to the importance of 2’-amino-2’-deoxynucleosides as potent therapeutics and reactants,
several paths for their synthesis have been developed. Aside from the synthesis methods
based on modifications of the monosaccharide molecule before coupling with the nucleobase,
there are three independent methods for the synthesis of 2’-amino-2’-deoxynucleosides
exploiting the transformation of uridine and its derivatives. The first one relies on substitution
of a leaving group by the phthalimide ion, and subsequent cleavage with hydrazine.10,24 In
another approach, 2,2’-anhydrouridine upon treatment with sodium azide, afforded the 2’-
azido-2’deoxy-derivative. Catalytic reduction of the azido group gave the desired 2’-
aminoderivative.25-28 As both of these reactions occur with inversion of configuration on the
atom bearing the leaving group the starting derivatives should possess the D-arabino
configuration, which in the course of reaction is inverted into D-ribo. In the third method the
3’-OH group of 2,2’-anhydrouridine, under treatment with an excess of trichloroacetonitrile,
afforded 3’-O-trichloroacetimidate, which under heating in the presence of catalytic amounts
of trimethylamine easily formed an intramolecular cyclic product, oxazoline.29,30 The
oxazoline when treated with ethanolic sodium hydroxide initially forms the 2’-N,3’-O-
oxazolidin-2-one intermediate, which on prolonged heating gave 2’-amino-2’-deoxyuridine in
70-80% yield.30
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In this report we present another approach involving the use of 5’-O-trityl-2,2’-
anhydrouridine, which under basic conditions, upon treatment with the appropriate
isothiocyanates (prepared from the methyl esters of amino acids or mono-protected α, ω-
diaminoalkanes), afforded the corresponding tetrahydrofuro[3,4-d]-oxazole-2(3H)-thione
derivatives. We explored two ways for the preparation of the final products. In the first one,
tetrahydrofuro[3,4-d]-oxazole-2(3H)-thiones were transformed by a ring opening reaction into
N-substituted 2’-amino-2’-deoxy-5’-O-trityluridine derivatives under basic conditions. In the
case of amino acid derivatives under applied conditions, the methyl ester group is also
hydrolysed. The opposite sequence of reactions was applied in the case of N-Boc derivatives;
the trityl protection was done prior to the oxazole-2(3H)-thione ring cleavage. In both cases
the yield of desired 2’-amino(substituted)-2’-deoxyuridine is satisfactory.
2. Results and discussion
The starting 2,2’-anhydrouridine 2 was prepared according to the known method31 avoiding to
use of HMPA. Commercially available uridine was reacted with diphenyl carbonate in the
presence of sodium bicarbonate in DMF solution at 80 oC. Reaction occurred smoothly (2 h to
complete) and 2 was isolated in 90% yield (Scheme 1). For the protection of the 5’-hydroxyl
group, the trityl group was selected due to its stability under basic conditions. Tritylation was
performed according to known reported method,32 with slight modification. The reaction was
carried out in a mixture of anhydrous DMSO and pyridine in the ratio 9:1 (v:v) at room
temperature. The 5’-O-trityl-2, 2’-anhydrouridine 3 was obtained in 73% yield. At the same
time in parallel experiments the second reactants, isothiocyanates derived from the
compounds possessing a primary amino group, were synthesized. There are several methods
for the transformation of primary amines into isothiocyanates. Usually carbon disulphide33-34
or thiophosgene35 is used as a source of thiocarbonyl group. As the amino group donors we
selected methyl esters of amino acids and α, ω-diaminoalkanes. In the latter, one of the amino
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groups was protected as the N-Boc derivative. Initially amino acids were reacted with
methanol in the presence of thionyl chloride giving the methyl esters as their hydrochlorides
in the crystal form.36 Mono protected NH-Boc α, ω-diaminoalkanes were received from
commercial sources.
Scheme 1. Synthesis of 5’-O-trityl-2,2’-anhydrouridine
The preparation of isothiocyanates was performed in a two-phase system. A suspension of
the selected amino compound 4a-g in chloroform and an aqueous saturated solution of
sodium bicarbonate, was treated with an excess of thiophosgene (1.5 eq.) at room temperature
(Scheme 2). The progress of reaction was monitored by TLC (30% EtOAc: n-hexane) and
after disappearance of the amino compounds the reaction mixture was diluted with cold water.
Scheme 2. Synthesis of isothiocyanates derived from amino compounds
Work up and purification on silica gel gave the appropriate isothiocyanates 5a-g in yields of
58%-75% and with sufficient purity for the next step. The reaction of 2, 2’-anhydrouridine 3
with isothiocyanates 5a-g was carried out in a solution of acetonitrile in the presence of 1,8-
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diazabicyclo[5.4.0]undec-7-ene (DBU) as a base (Scheme 3). The isothiocyanates and base
were used in a twofold excess with respect to anhydrouridine 3. The reaction was carried out
at room temperature and finished after disappearance of the uridine substrate (TLC, 5%
MeOH:CHCl3).
Scheme 3. Synthesis of 2’-(N-substituted)amino-2’-deoxyuridine derivatives
Purification using column chromatography (20% MeOH:CH2Cl2) gave the desired
tetrahydrofuro[3, 4-d]-oxazole-2(3H)-thione derivatives 6a-g in yields of 54-87%. The key
step of the synthetic pathway involved cleavage of the [3, 4-d]-oxazole-2(3H)-thione ring in
uridine derivative 6. In the case of compounds 6a-d, having a methyl alkanoate back bone, the
oxazole-2(3H)-thione ring was easily opened when treated with an aqueous solution of
sodium hydroxide at elevated temperature. Under these conditions the ester group present on
the 2’-N-alkyl terminal position was also cleaved and 7 were obtained in satisfactory yields of
65-73%. Deprotection of the 5’-hydroxyl group was carried out at room temperature under
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acidic conditions giving the final products 8a-d in good yield. For compounds 6e-g we
explored another approach for the preparation of the final products. According to our
observation the oxazole-2(3H)-thione ring in 6 is resistant under acidic conditions at room
temperature. This fact permits removal of both protecting groups, carbamate present in the N-
Boc fragment of 6, and the 5’-O-trityl ether simultaneously. When 6e-g was treated with
hydrochloric acid in acetonitrile solution at room temperature, both N-Boc and trityl
protecting groups were removed and 9a-c derivatives were isolated in good yield. The final
products 8e-g were obtained by oxazole-2(3H)-thione ring opening in the presence of base in
yields of 88% - 92% (Scheme 3).
O
N
O
N
O
N
O
N
N
O
N
O
N
O
NH
O
RNCS
TrO
O
S
O
O
O
O
O
DBU
TrO
TrO
TrO
DBUH
- DBU
MeCN, DBU
r.t
-DBUH
O
N
R
HO
NHR
NR
3
S
6
S
Scheme 4. Formation of tetrahydrofuro[3, 4-d]-oxazole-2(3H)-thione derivatives
Scheme 5. Introduction of fluorophore unit into 2’-aminofunctionalized uridine
The formation of 6 can be explained as a result of reaction sequences initiated by the reaction
of the 3’-OH group of the anhydrouridine with isothiocyanate. The initially formed
thiocarbamate is deprotonated by DBU resulting in intramolecular nucleophilic attack on the
carbon C-2’ of anhydrouridine. The erythro configuration of the 3’-OH group in 3 determines
the side of attack by the deprotonated thiocarbamate unit and subsequent formation of the
[3,4-d]-oxazole-2(3H)-thione ring (Scheme 4).
A
similar intermediate, namely
6

ACCEPTED MANUSCRIPT
tetrahydrofuro[3,4-d]oxazol-2(3H)-one was postulated in the reaction of 2,2’-anhydrouridine
with O-benzyl hydroxylamine in the presence of N,N’-carbonyldiimidazole.29 The resulting
3’-O-(benzyloxyamino)carbonyl derivative was converted into the corresponding 2’-
(benzyloxyamino)-2’-N,3’-O-carbonyl derivative under treatment with catalytic amounts of
DBU.
Finally we explored the usefulness of the obtained uridine derivatives 8 in the synthesis of
conjugates with a common fluorophore (Scheme 5).37,38 Thus 8g reacted with
benzo[de]isochromene-1,3-dione 10a and its 6-bromo derivative 10b at elevated temperature
to give the corresponding imide conjugates 11 in very good yield.
3. Conclusion
We have devised a new approach for the synthesis of 2’-amino-2’-deoxyuridine
derivatives where the amino group bears a linker with an additional functional group located
on the terminal carbon atom, either a carboxylic or amino group. By the choice of the order of
deprotection steps is possible to selectively remove the trityl and N-Boc protecting groups and
retain the oxazolidine-2-thione ring. Under basic conditions the oxazolidine-2-thione ring is
cleaved together with the ester group present in the 2’-NH-linker, whereas the 5’-O-trityl and
N-Boc groups are retained. The terminal functional group can be used for further
functionalization of the uridine e.g. conjugation with a fluorophore unit.
4. Experimental
4.1.General
All reagents and solvents were of analytical grade, obtained from commercial suppliers and
used without further purification except for CH2Cl2, which was distilled prior to use and
MeCN purified by distillation from P2O5 and dried through storage over activated 3A
molecular sieves. Anhydrous pyridine was dried over KOH pellets. Anhydrous Et3N was
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dried through storage over activated 4A molecular sieves. All reactions were monitored by
TLC using silica-gel-coated aluminium plates with a fluorescence indicator (SiO2 60, F254)
and were visualized by UV light. Column chromatography was performed using silica gel
packed columns (particle size 0.040-0.063 mm, Merck). 1H NMR spectra were recorded on a
Varian 600 MHz System or Agilent 400 MHz Spectrometer; 13C NMR spectra were recorded
at 150 MHz or 100 MHz, respectively. Chemical shifts were measured relative to residual
non-deuterated solvent resonances. Melting points were determined using a Boethius M HMK
hot-stage apparatus. IR spectra were recorded on Nicolet 6700 FT-IR Spectrometer (Thermo
Scientific). High-resolution electrospray ionisation mass spectroscopy (ESI-MS) experiments
were performed using a Waters Xevo G2 QTOF instrument equipped with an injection system
(cone voltage 50 V; source 120 °C).
2, 2’-Anhydrouridine 231 and 5’-O-trityl-2, 2’anhydrouridine 332 was obtained in yields of
89% and 75%, respectively. Methyl esters of selected amino acids were prepared according to
literature data.36 N-Boc-1,4-butanediamine and N-Boc-1,6-hexanediamine were purchased
from Sigma-Aldrich.
4.2. Typical procedure for preparation of isothiocyanates 5a-g
To a mixture of sodium bicarbonate solution (10 mL, sat. aq.) and CH2Cl2 (10 mL) the
hydrochloride of amino acid methyl ester 5a-d or N-Boc-mono-protected α,ω-diaminoalkane
o
(15mmol) 4e-g was added. The reaction mixture was cooled down to 0 C and a solution of
thiophosgene (15 mmol) in CH2Cl2 (5mL) was added dropwise while stirring. After 30-60
min. TLC indicated a total disappearance of substrate (TLC, 30% EtOAc/n-hexane). The
organic layer was separated and the aqueous layer was extracted with CH2Cl2 (3x10 mL). The
organic extracts were collected together, dried (Na2SO4) and the solvent was evaporated under
diminished pressure. The residual oil was purified on silica gel (30% EtOAc/n-hexane) and
afforded the desired isothiocyanates as viscous yellow oils.
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4.2.1. Methyl 2-isothiocyanatoacetate 5a.39 Yield 58% (1.24 g); δH (600 MHz, CDCl3) 3.84 (s,
2H), 4.26 (s, 3H); δC (150 MHz, CDCl3) 166.6 (C=O), 138.4 (C=S), 53.2, 46.3; MS (ESI)
[M+H]+ calcd for C4H6NO2S=132.0119, Found 132.0126; Rf (30% EtOAc/n-C6H14) 0.52.
4.2.2. Methyl 3-isothiocyanatopropanoate 5b.39 Yield 59% (1.28 g); δH (600 MHz, CDCl3)
2.71 (t, 6.6 Hz, 2H), 3.75 (s, 3H), 3.82 (t, J 6.6 Hz, 2H); δC (150 MHz, CDCl3)170.3, 126.9,
52.2, 40.8, 34.4; MS (ESI) [M+H]+ calcd for C5H8NO2S=146.0276, Found=146.0281; Rf
(30% EtOAc/n-C6H14) 0.49.
4.2.3. Methyl 4-isothiocyanatobutanoate 5c.40 Yield 75% (1.79 g); δH (600 MHz, CDCl3)
2.00-2.05 (m, 2H), 2.48 (t, J 7.2 Hz, 2H), 3.64 (t, J 6.6 Hz, 2H), 3.70 (s, 3H); δC (150 MHz,
CDCl3) 172.5, 130.5, 51.7, 44.3, 30.5, 25.1; MS (ESI) [M+Na]+ calcd for C6H9NO2SNa=
182.0252, Found=182.0250; Rf (30% EtOAc/n-C6H14) 0.47.
4.2.4. Methyl 6-isothiocyanatohexanoate 5d. Yield 71% (1.99 g); FT IR [ATR,ν cm-1] 2048,
1746, 436, 1208, 1150, 1054; δH (600 MHz, CDCl3) 1.43-1.48 (m, 2H), 1.65-1.75 (m, 4H),
2.40 (t, J 7.8 Hz, 2H), 3.53 (t, J 6.6 Hz, 2H), 3.68 (s, 3H); δC (150 MHz, CDCl3) 173.7, 128.0,
51.6, 44.9, 33.7, 26.1, 26.0, 24.1; MS (ESI) [M+Na]+ calcd for C8H13NO2SNa=210.2491,
Found=210.2490; Rf (30% EtOAc/n-C6H14) 0.46.
4.2.5. t-Butyl 2-isothiocyanatoethylcarbamate 5e.41 Yield 63% (1.91g), white solid; Mp 63-64
o
oC [65 C]41; FT IR [ATR,
ν
cm-1] 2932, 2095, 1687, 1510, 1165; δH (400 MHz, CDCl3) 1.46
(s, 9H), 3.41-3.36 (m, 2H), 3.65 (t, J 5.7 Hz, 2H), 4.93 (br. s, 1H); δC (100 MHz, CDCl3)
155.6, 132.3, 80.1, 45.4, 40.8, 28.3; MS (ESI) [M+ H]+ calcd for C8H15N2O2S=203.0854,
Found=203.0850; Rf (5% MeOH/CHCl3) 0.56.
4.2.6. t-Butyl 4-isothiocyanatobutylcarbamate 5f.42 Yield 60% (2.1 g) white semisolid; δH
(600 MHz, CDCl3) 1.45 (s, 9H), 1.58-1.65 (m, 2H), 1.70-1.77 (m, 2H), 3.16 (q, J 9.6 Hz, 2H),
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3.59 (t, J 9.6 Hz, 2H), 4.63 (br s, 1H); δC (150 MHz, CDCl3) 155.6, 132.3, 80.1, 45.4, 40.8;
MS (ESI) [M+ H]+ calcd for C10H19N2O2S=231.1167, Found=231.1158; Rf (5%
MeOH/CHCl3) 0.62.
4.2.7. t-Butyl 6-isothiocyanatohexylcarbamate 5g.42 Yield 72% (2.8 g) white semisolid; δH
(600 MHz, CDCl3) 1.31-1.54 (m, 15H), 1.67-1.74 (m, 2H), 3.13 (q, J 6.4 Hz, 2H), 3.51 (t, J
6.4 Hz, 2H), 4.59 (br s, 1H); δC (150 MHz, CDCl3) 155.9, 129.8, 79.0, 44.9, 40.3, 29.8, 29.8,
28.8, 26.2, 25.9; MS (ESI) [M+H]+ calcd for C12H23N2O2S=259.1480, Found=259.1493; Rf
(5% MeOH/CHCl3) 0.74.
4.3. Typical procedure for preparation of compounds 6a-g
To a suspension of 5'-O-trityl-2,2'-anhydro-β-D-uridine 3 (1.7 mmol) in anhydrous MeCN (5
mL), isothiocyanates 5a-g (3.4 mmol) and DBU (3.4 mmol) were added under argon. After
completion of the reaction (TLC, 5% MeOH/CHCl3) the reaction mixture was washed with an
aqueous solution of citric acid (10%, 2 x 10 mL). The aqueous layer was extracted with
EtOAc (3x5 mL). The combined organic layers were washed with H2O (5 mL), dried
(Na2SO4) and concentrated. Purification of the crude product by column chromatography (5%
MeOH/CHCl3) gave the compound 6a-6g as a white solid.
4.3.1. Methyl 2-((3aR,4R,6R,6aS)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-thioxo-6-
(trityloxymethyl)tetrahydrofuro[3,4-d]oxazol-3(2H)-yl)acetate 6a. Yield 87% (0.89 g); Mp
o
147-149 C; FT IR [ATR,
ν
cm-1] 2953, 1679, 1541, 1448, 1251, 1208, 1000; δH (600 MHz,
CDCl3) 3.52-3.54 (dd, J 11.4, 3.0 Hz, 1H), 3.60-3.63 (dd, J 11.4, 4.2 Hz, 1H), 3.70 (s, 3H),
3.89-3.95 (m, 2H), 4.56-4.62 (m, 1H), 4.83-4.86 (m, 1H), 5.41 (d, J 7.8 Hz, 1H), 5.45-5.46
(m, 1H), 6.06 (d, J 1.8 Hz, 1H), 7.27-7.36 (m, 15H), 7.67 (d, J 7.8 Hz, 1H), 9.25 (s, 1H); δC
(150 MHz, CDCl3) 188.0, 168.3, 163.1, 150.32, 142.7, 139.2, 128.5, 128.2, 127.7, 102.7,
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90.5, 87.9, 86.2, 80.9, 71.6, 62.1, 52.7, 48.4; MS (ESI) [M+H]+ calcd for C32H30N3O7S=
600.1804, Found =600.1802; Rf (5% MeOH/CHCl3) 0.40.
4.3.2. Methyl 3-((3aR,4R,6R,6aS)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-thioxo-6-
(trityloxymethyl)tetrahydrofuro[3,4-d]oxazol-3(2H)-yl)propanoate 6b. Yield 54% (0.56 g);
o
Mp 106-107 C; FT IR [ATR,
ν
cm-1] 2925, 1683, 1447, 1258, 1202, 1001; δH (600 MHz,
CDCl3) 2.73-2.77 (m, 2H), 3.05-3.30 (m, 1H), 3.53-3.62 (m, 1H), 3.69 (s, 3H), 3.91-3.96 (m,
1H), 4.22-4.26 (m, 1H), 4.43-4.45 (m, 1H), 4.94-4.96 (m, 1H), 5.40 (d, J 7.8 Hz, 1H), 5.44-
5.46 (m, 1H), 6.12 (d, J 1.8 Hz, 1H), 7.28-7.38 (m, 15H), 7.67 (d, J 7.8 Hz, 1H), 9.23 (s, 1H);
δC (150 MHz, CDCl3)186.6, 172.9, 163.0, 150.2, 143.0, 139.9, 128.7, 128.30, 128.8, 103.4,
90.4, 87.9, 85.6, 80.9, 71.2, 62.5, 52.3, 42.7, 31.5; MS (ESI) [M+H]+ calcd for C33H32N3O7S=
614.1961, Found=614.1960; Rf (5% MeOH/CHCl3) 0.45.
4.3.3. Methyl 4-((3aR,4R,6R,6aS)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-thioxo-6-
(trityloxymethyl)tetrahydrofuro[3,4-d]oxazol-3(2H)-yl)butanoate 6c. Yield 69% (0.73 g); FT
IR [ATR,ν
cm-1] 2927, 1683, 1446, 1256, 1201, 1003; δH (600 MHz, CDCl3) 2.00-2.05 (m,
2H), 2.43-2.46 (m, 2H), 3.52 (dd, J 10.8, 3.6 Hz, 1H), 3.60 (dd, J 10.8, 3.0 Hz, 1H), 3.66 (s,
3H), 4.10-4.17 (m, 2H), 4.43-4.45 (m, 1H), 4.75 (dd, J 9.0, 1.8 Hz, 1H), 5.38 (dd, J 9.0, 4.8
Hz, 1H), 5.42 (dd, J 8.4, 2.4 Hz, 1H), 6.05 (d, J 2.4 Hz, 1H), 7.26-7.30 (m, 15H), 7.69 (d, J
7.8 Hz, 1H), 9.45 (s, 1H); δC (150 MHz, CDCl3) 186.8, 173.7, 163.1, 150.2, 142.9, 139.9,
128.7, 128.3, 127.8, 103.2, 90.3, 87.9, 86.1, 80.6, 70.3, 62.4, 52.0, 46.5, 31.1, 21.4; MS (ESI)
[M+H]+ calcd for C34H34N3O7S =628.2117, Found=628.2115; Rf (5% MeHO/CHCl3) 0.64.
4.3.4. Methyl 6-((3aR,4R,6R,6aS)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-thioxo-6-
(trityloxymethyl)tetrahydrofuro[3,4-d]oxazol-3(2H)-yl)hexanoate 6d. Yield 81% (0.89 g); Mp
o
65-66 C; FT IR [ATR,
ν
cm-1] 2929, 1685, 1448, 1255, 1207, 1006; δH (600 MHz, CDCl3)
1.39-1.40 (m, 2H), 1.65-1.69 (m, 4H), 2.31-2.34 (m, 2H), 3.55-3.60 (m, 4H), 3.65 (s, 3H),
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4.42-4.44 (m, 1H), 4.62-4.64 (dd, J 9.0, 2.4 Hz, 1H), 5.39-5.40 (m, 2H), 6.06-6.07 (d, J 1.8
Hz, 1H), 7.27-7.37 (m, 15H), 7.70 (d, J 1.8 Hz, 1H), 9.55 (s, 1H); δC (150 MHz, CDCl3)
186.5, 174.0, 163.0, 150.1, 142.8, 139.6, 128.6, 128.2, 127.7, 103.2, 89.8, 87.9, 86.0, 79.9,
70.5, 62.17, 51.6, 47.0, 33.8, 26.0, 25.6, 24.4; MS (ESI) [M+H]+ calcd for C36H38N3O7S=
656.2430, Found = 656.2430; Rf (5% MeOH/CHCl3) 0.65.
4.3.5. t-Butyl 2-((3aR,4R,6R,6aS)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-thioxo-6-
(trityloxymethyl)tetrahydrofuro[3,4-d]oxazol-3(2H)-yl)ethylcarbamate 6e. Yield 65% (0.74
g); FT IR [ATR,ν
cm-1] 2934, 1686, 1449, 1254, 1159; δH (400 MHz, DMSO-d6) 1.34 (s, 9H),
3.17-3.22 (m, 2H), 3.26 (dd, J 10.4, 4.4 Hz, 1H), 3.31-3.33 (br, 1H), 3.40-3.47 (m, 2H), 3.98-
4.04 (m, 1H), 4.30 (dt, J 6.4, 4.4 Hz, 1H), 5.02 (d, J 8.0 Hz, 1H), 5.26 (dd, J 8.8, 4.8 Hz, 1H),
5.60 (dd, J 8.0, 2.0 Hz, 1H), 6.00 (d, J 2.4 Hz, 1H), 6.94 (t, J 6.0 Hz, 1H), 7.20 (d, J 8.0 Hz,
1H), 7.25-7.40 (m, 15H); δC (100 MHz, DMSO-d6) 186.2, 163.1, 155.6, 150.1, 143.3, 142.2,
128.2, 127.9, 127.1, 102.1, 91.1, 86.3, 85.1, 82.2, 77.9, 68.3, 63.4, 46.3, 36.5, 28.1; HRMS
(ESI) [M+H]+ calcd for C36H39N4O7S=671.2539, Found=671.2534; Rf (5% MeOH/CH2Cl2)
0.65.
4.3.6. t-Butyl 4-((3aR,4R,6R,6aS)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-thioxo-6-
(trityloxymethyl)tetrahydrofuro[3,4-d]oxazol-3(2H)-yl)butylcarbamate 6f. Yield 87% (1.03
o
g); Mp 109-110 C; FT IR [ATR,
ν
cm-1] 2938, 1684, 1446, 1258, 1157,740; δH (600 MHz,
DMSO-d6) 1.36-1.39 (m, 11H), 1.53-1.63 (m, 2H), 2.88-2.97 (m, 2H), 3.28 (dd, J 10.2, 4.2
Hz, 1H), 3.42 (dd, J 10.2, 6.6 Hz, 1H), 3.83-3.88 (m, 2H), 4.30 (dt, J 6.6, 4.8 Hz, 1H),
4.97(dd, J 8.4, 2.4 Hz, 1H), 5.32 (dd, J 8.4, 4.8 Hz, 1H), 5.57 (d, J 7.8 Hz, 1H), 6.00 (d, J 1.8
Hz, 1H), 6.78 (t, J 5.4 Hz, 1H), 7.28-7.40 (m, 15H), 7.72 (d, J 7.8 Hz, 1H), 11.46 (s, 1H); δC
(150 MHz, DMSO-d6) 185.6, 163.0, 155.5, 150.2, 143.2, 141.9, 128.1, 127.9, 127.1, 101.9,
12

ACCEPTED MANUSCRIPT
90.7, 86.3, 85.2, 81.6, 77.3, 68.1, 63.2, 46.0, 40.0, 28.2, 26.7, 22.5; MS (ESI) [M+H]+ calcd
for C38H43N4O7S=698.2774, Found=698.2770; Rf (5% MeOH/CH2Cl2) 0.43.
4.3.7. t-Butyl 6-((3aR,4R,6R,6aS)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-thioxo-6-
(trityloxymethyl)tetrahydrofuro[3,4-d]oxazol-3(2H)-yl)hexylcarbamate 6g. Yield 76% (0.82
o
g); Mp 89-90 C; FT IR [ATR,
ν
cm-1] 2936, 1686, 1447, 1256, 1159, 748; δH (600 MHz,
DMSO-d6) 1.22-1.26 (m, 4H), 1.34-1.37 (m, 11H), 1.53-1.63 (m, 2H), 2.88 (dd, J 13.6, 6.6
Hz, 2H), 3.27 (dd, J 10.4, 4,1 Hz, 1H), 3.35 (dd, J 10.4, 6.6 Hz, 1H), 3.43-3.47 (m, 1H), 3.84
(ddd, J 13.8, 9.5, 6.8 Hz, 1H), 4.30 (dd, J 6.6, 4.1 Hz, 1H), 4.96 (dd, J 8.4, 2.4 Hz, 1H), 5.30
(dd, J 8.4, 4.1 Hz, 1H), 5.57 (dd, J 7.8, 2.2 Hz, 1H), 6.00 (d, J 2.2 Hz, 1H), 6.73 (t, J 5.4 Hz,
1H), 7.27-7.40 (m, 15H), 7.70 (d, J 7.8 Hz, 1H), 11.45 (d, J 2.2 Hz, 1H); δC (150 MHz,
DMSO-d6) 185.3, 163.1 , 155.6, 150.2, 143.3, 141.8, 128.2, 128.0, 127.2, 102.1, 90.5, 86.4,
85.0, 81.6, 77.3, 68.1, 63.2, 46.3, 39.8, 29.3, 28.3, 25.9, 25.8, 25.2; MS (ESI) [M+H]+ calcd
for C40H47N4O7S=727.3165, Found=727.3164; Rf (5% MeOH/CH2Cl2) 0.42.
4.4. Preparation of 2’-amino-2’-deoxyuridine derivatives 7a-d and 8e-g
Compound 6a-d or 9a-c (0.5 mmol) was dissolved in aqueous ethanol (10 mL EtOH, 2 mL
H2O) containing NaOH (10 mmol). The reaction mixture was refluxed for two hours (TLC,
10% MeOH:CHCl3). After disappearance of substrate, the reaction mixture was cooled down,
portioned between EtOAc (50 mL) and H2O (20 mL) and neutralized with 20% aq. HCl. The
organic layer was separated, and the aqueous solution was extracted with EtOAc (2 x 10 mL).
The organic extracts were combined, dried (MgSO4) and the volatiles were removed under
diminished pressure. Purification of the residue on silica gel using gradient MeOH (10% to
50% in CH2Cl2) gave the product 7a-d or 8e-g as a white semisolid.
4.4.1. 2-((2R, 3R, 4S, 5R)-2-(2, 4-Dioxo-3, 4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-
(trityloxymethyl)tetrahydrofuran-3-ylamino)ethanoic acid 7a. Yield 66% (0.18 g); FT IR
13

ACCEPTED MANUSCRIPT
[ATR,
ν
cm-1] 3058, 1686, 1245, 1112, 1078, 763; δH (600 MHz, DMSO-d6) 3.13-3.17 (m,
2H), 3.21-3.48 (m, 5H), 4.07 (d, J 3.0 Hz, 1H), 4.26-4.32 (m, 2H), 5.36 (d, J 8.0 Hz, 1H),
7.23-7.54 (m, 15H), 5.80 (s, 1H), 7.74 (d, J 8.0 Hz, 1H), 11.41 (s, 1H); δC (150 MHz, DMSO-
d6) 163.43, 151.06, 143.81, 140.77, 128.74, 128.44, 127.61, 101.88, 87.71, 86.92, 83.85,
74.07, 68.76, 64.11, 63.86, 55.33, 49.58; MS (ESI) [M+H]+ calcd for C30H30N3O7=544.2084,
Found=544.2082; Rf (10% MeOH/CH2Cl2) 0.22.
4.4.2. 3-((2R,3R,4S,5R)-2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(trityloxy
methyl)tetrahydrofuran-3-ylamino)propanoic acid 7b. Yield 65% (0.18 g); FT IR [ATR,ν
cm-
1] 3058, 1683, 1563, 1448, 1405, 1270, 1082, 772; δH (600 MHz, DMSO-d6) 2.14-2.31 (m,
4H), 2.72 (t, J 6.2 Hz, 2H), 3.16-3.22 (m, 3H), 3.32 (dd, J 10.5, 4.9 Hz, 1H), 3.99 (d, J 3.6 Hz,
1H), 4.20-4.26 (m, 2H), 5.36 (d, J 8.1 Hz, 1H), 7.23-7.46 (m, 15H), 7.66 (d, J 8.1 Hz, 1H),
11.45 (s, 1H); δC (150 MHz, DMSO-d6) 171.70, 170.79, 158.48, 151.24, 148.29, 136.13,
135.86, 135.04, 109.47, 94.86, 94.31, 91.45, 76.53, 71.80, 71.59, 52.00, 44.56; MS (ESI)
[M+H]+ calcd for C31H32N3O7=558.2240, Found=558.2238; Rf (10% MeOH/CH2Cl2) 0.26.
4.4.3. 4-((2R,3R,4S,5R)-2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(trityloxy
methyl)tetrahydrofuran-3-ylamino)butanoic acid 7c. Yield 73% (0.21 g); FT IR [ATR,ν
cm-1]
3058, 1686, 1557, 1447, 1404, 1270, 1082, 747; δH (600 MHz, DMSO-d6) 1.56-1.66 (m, 2H),
2.16-2.25 (m, 2H), 2.56-2.58 (m, 2H), 3.17-3.25 (m, 2H), 3.33 (dd, J 10.5, 4.8 Hz, 1H), 4.00
(dd, J 7.9, 4.0 Hz, 1H), 4.18 (dd, J 5.1, 4.4 Hz, 1H), 5.35 (d, J 8.1 Hz, 1H), 5.71 (d, J 6.2 Hz,
1H), 7.24-7.43 (m, 15H), 7.67 (d, J 8.1 Hz, 1H), 11.34 (s, 1H); δC (150 MHz, DMSO-d6)
162.8, 150.5, 143.3 140.3, 128.2, 127.9, 127.1, 101.5, 87.0, 86.4, 83.7, 68.2, 63.54, 63.2, 46.6,
31.7, 25.0; MS (ESI) [M+H]+ calcd for C32H34N3O7=572.2397, Found=572.2395; Rf (10%
MeOH/CH2Cl2) 0.26.
14

ACCEPTED MANUSCRIPT
4.4.4. 6-((2R, 3R, 4S, 5R)-2-(2, 4-Dioxo-3, 4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(trityl
oxymethyl)tetrahydrofuran-3-ylamino)hexanoic acid 7d. Yield 71% (0.21 g); FT IR [ATR,
ν
cm-1] 3059, 1687, 1549, 1447, 1270, 1083, 765; δH (600 MHz, DMSO-d6) 1.26 (dt, J 14.1, 7.1
Hz, 2H), 1.35-1.43 (m, 4H), 1.45-1.57 (m, 4H), 2.18 (t, J 7.3 Hz, 2H), 2.56 (dd, J 11.6, 5.2
Hz, 1H), 3.16-3.35 (m, 3H), 3.90-4.05 (m, 1H), 4.11-4.22 (m, 1H), 5.37 (d, J 8.0 Hz, 1H),
5.71 (d, J 5.2 Hz, 1H), 7.28-7.41 (m, 15H), 7.67 (d, J 8.0 Hz, 1H), 11.35 (s, 1H); δC (150
MHz, DMSO-d6) 174.34, 162.83, 150.54, 143.33, 140.35, 128.23, 127.93, 127.14, 101.55,
87.52, 86.42, 83.75, 68.22, 63.57, 47.11, 33.61, 26.15, 24.37; MS (ESI) [M+H]+ calcd for
C34H38N3O7=600.2710, Found=600.2708; Rf (10% MeOH/CH2Cl2) 0.31.
4.5. Preparation of 8a-d and 9a-c
Protected derivative of uridine 7a-d or 6e-g (0.6 mmol) was dissolved in CH2Cl2 (10 mL) and
treated with concentrated HCl (0.3 mL, d=1.18 g/mL, 3.3 mmol) at room temperature. After
completion of the reaction (TLC, 1% Et3N in 50%MeOH/CH2Cl2), H2O (10 mL) was added
and the reaction mixture was neutralized with saturated NaHCO3 solution. The water layer
was washed with CH2Cl2 (2 x 3 mL) and the organic layer discarded. The water was removed
under diminished pressure and the residue was dissolved in minimal amounts of MeOH and
purified by column chromatography (1% Et3N in 50%MeOH/CH2Cl2).
4.5.1. 2-((2R, 3R, 4S, 5R)-2-(2, 4-Dioxo-3, 4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(hydro
xylmethyl)tetrahydrofuran-3-ylamino)acetic acid 8a. Yield 93% (0.17 g) white semisolid; FT
IR [ATR,ν
cm-1] 3224, 1687, 1581, 1396, 1271, 1094, 814; δH (600 MHz, D2O) 3.84 (dd, J
12.7, 4.0 Hz, 1H), 3.90 (dd, J 12.7, 3.1 Hz, 1H), 3.92-3.96 (m, 1H), 4.00-4.05 (m, 1H), 4.24
(dd, J 6.7 Hz, 6.0 Hz, 1H), 4.31-4.34 (m, 1H), 4.67 (dd, J 6.0, 3.1 Hz, 1H), 5.97 (d, J 8.1 Hz,
1H), 6.37 (d, J 6.7 Hz, 1H), 7.92 (d, J 8.1 Hz, 1H); δC (150 MHz, D2O) 169.23, 165.95,
15

ACCEPTED MANUSCRIPT
151.78, 141.08, 103.06, 86.80, 85.66, 68.31, 62.06, 60.75, 47.22; MS (ESI) [M+H]+ calcd for
C11H16N3O7=302.0988, Found=302.0986; Rf (30% MeOH/CH2Cl2) 0.28.
4.5.2. 3-((2R,3R,4S,5R)-2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(hydroxy
methyl)tetrahydrofuran-3-ylamino)propanoic acid 8b. Yield 89% (0.17 g) white semisolid;
FT IR [ATR,ν
cm-1] 3229, 1682, 1575, 1396, 1273, 1107, 1052, 815; δH (600 MHz, D2O)
2.53-2.62 (m, 2H), 3.15-3.25 (m, 2H), 3.85 (dd, J 12.6, 4.2 Hz, 1H), 3.91 (dd, J 12.6, 3.6 Hz,
1H), 3.92-3.94 (m, 1H), 4.29-4.31 (m, 1H), 4.62 (dd, J 6.0, 3.0 Hz, 1H), 5.98 (d, J 8.4 Hz,
1H), 6.20 (d, J 6.6. Hz, 1H), 7.95 (d, J 8.4 Hz, 1H); δC (150 MHz, D2O) 170.3, 168.7, 154.5,
144.0, 105.5, 89.1, 71.1, 65.3, 63.6, 46.7, 36.6; MS (ESI) [M+H]+ calcd for
C12H18N3O7=316.1144, Found=316.1142; Rf (30% MeOH/CH2Cl2) 0.29.
4.5.3. 4-((2R,3R,4S,5R)-2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(hydroxy
methyl)tetrahydrofuran-3-ylamino)butanoic acid 8c. Yield 82% (0.16 g) white semisolid; FT
IR [ATR,ν
cm-1] 3240, 1674, 1545, 1409, 1271, 1104, 1050, 815; δH (600 MHz, D2O) 1.96
(dt, J 13.8, 7.0 Hz, 2H), 2.33 (t, J 7.0 Hz, 2H), 2.98-3.17 (m, 2H), 3.83 (dd, J 12.6, 4.2 Hz,
1H), 3.86 (dd, J 12.6, 3.6 Hz, 1H), 3.96-3.98 (t, J 6.4 Hz, 1H), 4.27 (dd, J 6.4, 3.1 Hz, 1H),
4.61 (dd, J 5.6, 2.8 Hz, 1H), 5.97 (d, J 8.1 Hz, 1H), 6.26 (d, J 7.1 Hz, 1H), 7.91 (d, J 8.1 Hz,
1H); δC (150 MHz, D2O) 184.4, 168.6, 154.4, 143.9, 105.7, 89.1, 88.3, 71.0, 64.6, 63.6, 49.6,
37.4, 25.7; MS (ESI) [M+H]+ calcd for C13H20N3O7=330.3137, Found=330.3135; Rf (30%
MeOH/CH2Cl2) 0.32.
4.5.4. 6-((2R, 3R, 4S, 5R)-2-(2, 4-Dioxo-3, 4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-
(hydroxy methyl)tetrahydrofuran-3-ylamino)hexanoic acid 8d. Yield 95% (0.20 g) white
semisolid; FT IR [ATR,ν
cm-1] 3259, 1686, 1448, 1271, 1079, 812; δH (600 MHz, D2O) 1.39-
1.41 (m, 2H), 1.58-1.64 (m, 2H), 1.72-1.80 (m, 2H), 2.21 (t, J 7.4 Hz, 2H), 3.10-3.19 (m, 2H),
3.85 (dd, J 12.6, 4.0 Hz, 2H),3.89 (dd, J 12.6, 3.2 Hz, 1H), 4.11 (dd, J 7.1, 6.0 Hz, 1H), 4.30-
16

ACCEPTED MANUSCRIPT
4.33 (m, 1H), 4.66 (dd, J 6.0, 2.6 Hz, 1H), 5.98 (d, J 8.1 Hz, 1H), 6.35 (d, J 7.1 Hz, 1H), 7.92
(d, J 8.1 Hz, 1H); δC (150 MHz, D2O) 182.8, 165.9, 151.7, 144.2, 103.2, 86.8, 85.4, 68.4,
61.8, 60.9, 47.0, 36.6, 25.5, 25.4, 25.0; MS (ESI) [M+H]+ calcd for C15H24N3O7= 358.1614,
Found: 358.1614; Rf (30% MeOH/CH2Cl2) 0.30.
4.5.5.
1-((2R,3R,4S,5R)-3-(2-Aminoethylamino)-4-hydroxy-5-(hydroxymethyl)tetrahydro
furan-2-yl)pyrimidine-2,4(1H,3H)-dione 8e. Yield 91% (0.15 g) white semisolid; FT IR
cm-1] 3348, 1680, 1566, 1387, 1261; δH (600 MHz, D2O) 2.89-2.95 (m, 2H), 3.28-
[ATR,
ν
3.33 (m, 2H), 3.84 (dd, J 12.6, 4.2 Hz, 1H), 3.90 (dd, J 12.6, 3.6 Hz, 1H), 3.95-3.98 (m, 2H),
4.27-4.30 (m, 2H), 4.60 (dd, J 6.0, 3.0 Hz, 1H), 5.97 (d, J 8.4 Hz, 1H), 6.22 (d, J 6.6. Hz, 1H),
7.98 (d, J 8.4 Hz, 1H); δC (150 MHz, D2O) 169.8, 166.7 , 153.2, 143.7, 105.2, 89.0, 712.0,
65.1, 63.2, 48.7, 43.6; MS (ESI) [M+H]+ Calcd for C11H19N4O5 =287.1355, Found=287.1355;
Rf (40% MeOH/CH2Cl2) 0.20.
4.5.6.
1-((2R,3R,4S,5R)-3-(4-Aminobutylamino)-4-hydroxy-5-(hydroxymethyl)tetrahydro
furan-2-yl)pyrimidine-2,4(1H,3H)-dione 8f. Yield 88% (0.17 g) white semisolid; FT IR
cm-1] 3346, 1678, 1565, 1384, 1260; δH (600 MHz, D2O) 1.38-1.44 (m, 2H), 1.54-
[ATR,
ν
1.58 (m, 2H), 2.61 (t, J 7.2 Hz, 2H), 3.03-3.11 (m, 2H), 3.83 (dd, J 12.6, 4.2 Hz, 1H), 3.88
(dd, J 12.6, 4.2 Hz, 1H), 3.96-3.97 (m, 2H), 4.28-4.29 (m, 2H), 4.61-4.64 (m, 1H), 5.97 (d, J
8.4 Hz, 1H), 6.24 (d, J 7.2 Hz, 1H), 7.92 (d, J 8.4 Hz, 1H); δC (150 MHz, D2O) 168.5, 156.0,
144.1, 105.8, 89.6, 88.4, 71.2, 65.0, 63.9, 47.7, 44.2, 36.1, 26.7; MS (ESI) [M+H]+ calcd for
C13H23N4O5=315.1668, Found=315.1669; Rf (40% MeOH/CH2Cl2) 0.22.
4.5.7.
1-((2R,3R,4S,5R)-3-(6-Aminohexylamino)-4-hydroxy-5-(hydroxymethyl)tetrahydro
furan-2-yl)pyrimidine-2,4(1H,3H)-dione 8g. Yield 92% (0.19 g) white semisolid; FT IR
cm-1] 3340, 1674, 1561, 1388, 1264; δH (600 MHz, D2O) 1.32-1.39 (m, 2H), 1.42-
[ATR,
ν
1.46 (m, 2H), 1.62-1.66 (m, 4H), 2.58-2.62 (m, 2H), 3.05-3.12 (m, 2H), 3.80 (dd, J 12.6, 4.2
17

ACCEPTED MANUSCRIPT
Hz, 1H), 3.86 (dd, J 12.6, 4.2 Hz, 1H), 3.92 (dd, J 12.6, 3.0 Hz, 1H), 4.28-4.31 (m, 2H), 4.61
(dd, J 6.0, 3.0 Hz, 1H), 5.95 (d, J 7.8 Hz, 1H), 6.23 (d, J 7.2 Hz, 1H), 7.92 (d, J 7.8 Hz, 1H);
δC (150 MHz, D2O)168.7, 154.5, 144.2, 105.7, 89.4, 88.3, 71.2, 64.9, 63.8, 46.1, 44.9, 29.4,
27.5, 26.5, 26.2; MS (ESI) [M+H]+ calcd for C15H27N4O5=343.1981, Found=343.1979; Rf
(40% MeOH/CH2Cl2) 0.25.
4.5.8. 1-((3aR,4R,6R,6aS)-3-(2-Aminoethyl)-6-(hydroxymethyl)-2-thioxohexahydrofuro[3,4-
d]oxazol-4-yl)pyrimidine-2,4(1H,3H)-dione 9a. Yield 86% (0.17 g) white semisolid; FT IR
[ATR,ν
cm-1] 2938, 1682, 1631, 1489, 1447, 1266, 1102, 997; δH (600 MHz, DMSO) 2.69-
2.75 (m, 1H), 2.78-2.83 (m, 1H), 3.50-3.56 (m, 3H), 3.65 (dd, J 11.4, 4.8 Hz, 1H), 3.68 (dd, J
11.4, 4.8 Hz, 1H), 3.72 (ddd, J 14.4, 7.8, 6.6 Hz, 1H), 4.06 (ddd, J 14.4, 8.4, 6.0 Hz, 1H), 4.18
(dd, J 9.0, 4.2 Hz, 1H), 4.93 (dd, J 9.0, 3.0 Hz, 1H), 5.25 (dd, J 9.0, 4.2 Hz, 1H), 5.69 (d, J 7.8
Hz, 1H), 6.00 (d, J 3.0 Hz, 1H), 7.76 (d, J 7.8 Hz, 1H), 11.46 (d, J 1.8 Hz, 1H); δC (150 MHz,
DMSO) 185.9, 171.4, 163.4, 150.4, 141.7, 101.9, 90.5, 86.5, 82.2, 68.8, 60.5, 51.6, 42.2, 30.2;
MS (ESI) [M+H]+ calcd for C12H17N4O5S=329.0919, Found=329.0918; Rf (20%
MeOH/CH2Cl2) 0.10.
4.5.9. 1-((3aR,4R,6R,6aS)-3-(4-Aminobutyl)-6-(hydroxymethyl)-2-thioxohexahydrofuro[3,4-
d]oxazol-4-yl)pyrimidine-2,4(1H,3H)-dione 9b. Yield 89% (0.19 g) white solid; Mp 113-
114 °C; FT IR [ATR,ν
cm-1] 2933, 1683, 1629, 1489, 1448, 1266, 1102, 998; δH (600 MHz,
DMSO-d6) 1.32 (qn, J 7.2 Hz, 2H), 1.52-1.56 (m, 4H), 2.53-2.55 (m, 3H), 3.49 (ddd, J 13.8,
9.0, 4.8 Hz, 1H), 3.66 (dd, J 12.0, 4.8 Hz, 1H), 3.69 (dd, J 12.0, 4.8 Hz, 1H), 3.86 (ddd, J
13.8, 9.6, 6.6 Hz, 1H), 4.19 (dd, J 9.0, 4.8 Hz, 1H), 4.44 (s, 1H), 4.92 (dd, J 9.0, 2.4 Hz, 1H),
5.27 (dd, J 8.4, 4.2 Hz, 1H), 5.67 (d, J 7.8 Hz, 1H), 5.97 (d, J 2.4 Hz, 1H), 7.76 (d, J 7.8 Hz,
1H); δC (150 MHz, DMSO-d6) 185.8, 163.6, 150.6, 141.3, 102.1, 90.2, 86.6, 81.8, 68.3, 60.5,
18

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46.4, 40.9, 29.8, 22.9; MS (ES) [M+H]+ calcd for C14H21N4O5S= 357.1232, Found=357.1230;
Rf (20% MeOH/CH2Cl2) 0.12.
4.5.10. 1-((3aR,4R,6R,6aS)-3-(6-Aminohexyl)-6-(hydroxymethyl)-2-thioxohexahydrofuro[3,4-
d]oxazol-4-yl)pyrimidine-2,4(1H,3H)-dione 9c. Yield 85% (0.20 g) white solid; Mp: 99-
100 °C; FT IR [ATR,ν
cm-1] 2934, 1682, 1632, 1489, 1449, 1261, 1100, 998; δH (600 MHz,
DMSO-d6) 1.22-1.31 (m, 6H), 1.42 (qn, J 7.2 Hz, 2H), 1.51-1.64 (m, 4H), 2.63 (t, J 7.2 Hz,
2H), 3.48 (ddd, J 13.8, 9.0, 4.8 Hz, 1H), 3.66 (dd, J 12.0, 4.8 Hz, 1H), 3.70 (dd, J 12.0, 4.8
Hz, 1H), 3.85 (ddd, J 13.8, 9.6, 6.6 Hz, 1H), 4.19 (dd, J 9.0, 4.8 Hz, 1H), 4.91 (dd, J 8.4, 2.4,
1H), 5.29 (dd, J 8.4, 4.2 Hz, 1H), 5.68 (d, J 7.8 Hz, 1H), 5.99 (d, J 2.4 Hz, 1H), 7.78 (d, J 7.8
Hz, 1H); δC (150 MHz, DMSO) 185.6, 163.6, 150.6, 141.0, 102.0, 90.0, 86.4, 81.7, 68.3, 60.3,
46.3, 45.6, 29.9, 25.7, 25.6, 25.1; MS (ESI) [M+H]+ calcd for C16H25N4O5S= 385.1545,
Found=385.1544; Rf (20% MeOH/CH2Cl2) 0.13.
4.6. Conjugation with anhydride of 1, 8-naphthalenedicarboxylic acid
A mixture of benzo[de]isochromene-1,3-dione 10a, b (0.3 mmol) and 8g (0.1 g, 0.26 mmol)
in anhydrous ethanol (5 mL) containing pyridine (0.2 mL, 2.5 mmol) and 4-N,N-dimethyl-
pyridine (0.1g, 0.8 mmol) was refluxed under argon for 18 h and afterwards evaporated to
dryness. The traces of pyridine were removed by co-evaporation with anhydrous toluene (2 x
5 mL). The residual oil was purified on silica gel packed column using a mixture of 10%
MeOH/CHCl3 as an eluent.
4.6.1.
2-(6-((2R,3R,4S,5R)-2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-
(hydroxymethyl)tetrahydrofuran-3-ylamino)hexyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
cm-1] 3382, 2941,
11a. Yield 93% (0.13 g) yellowish solid; Mp 176-177 ºC; FT IR [ATR,
ν
1690, 1652, 1589, 1345, 1264, 1238, 1054, 779; δH (600 MHz, DMSO-d6) 1.22 (s, 1H), 1.29-
1.35 (m, 2H), 1.35-1.43 (m, 2H), 1.58-1.66 (m, 2H), 2.47-2.52 (m, 2H), 2.53-2.59 (m, 1H),
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3.17 (dd, J 6.5, 5.7 Hz, 1H), 3.53-3.62 (m, 2H), 3.87 (dd, J 6.4, 3.4 Hz, 1H), 3.99-4.06 (m,
2H), 4.11 (dd, J 5.2, 2.7 Hz, 1H), 5.08 (t, J 4.9 Hz, 1H), 5.42 (s, 1H), 5.65 (d, J 8.0 Hz, 1H),
5.72 (d, J 7.1 Hz, 1H), 7.87 (dd, J 8.2, 7.2 Hz, 3H), 7.91 (d, J 8.1 Hz, 1H), 8.45 (dd, J 8.4, 1.0
Hz, 2H), 8.49 (dd, J 7.3, 1.1 Hz, 2H), 11.29 (s, 1H); δC (150 MHz, DMSO-d6) 163.3, 162.9,
150.7, 140.6, 134.2, 131.2, 130.6, 127.2, 127.1, 122.0, 101.7, 86.6, 85.9, 68.4, 63.6, 61.1,
47.1, 39.5, 29.4, 27.4, 26.3, 26.2; MS (ESI) [M+H]+ calcd for C27H31N4O7= 523.2192,
Found=523.2190; Rf (10% MeOH/CHCl3) 0.15.
4.6.2. 6-Bromo-2-(6-((2R,3R,4S,5R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-
5-(hydroxymethyl)tetrahydrofuran-3-ylamino)hexyl)-1H-benzo[de]isoquinoline-1,3(2H)-
o
dione 11b. Yield 97% (0.15 g) yellowish solid; Mp 109-110 C; FT IR [ATR,
ν
cm-1] 3391,
2940, 1694, 1622, 1589, 1346, 1264, 1238, 1054, 779; δH (600 MHz, DMSO-d6) 1.22 (s, 1H),
1.25-1.35 (m, 2H), 1.35-1.42 (m, 2H), 1.61 (dt, J 14.6, 7.3 Hz, 2H), 2.47-2.53 (m, 2H), 2.53-
2.59 (m, 1H), 3.13-3.21 (m, 1H), 3.55 (dd, J 11.9, 3.2 Hz, 1H), 3.59 (dd, J 11.9, 3.7 Hz, 1H),
3.87 (dd, J 6.3, 3.4 Hz, 1H), 3.97-4.04 (m, 2H), 4.11 (dd, J 5.1, 2.7 Hz, 1H), 5.08 (s, 1H), 5.42
(s, 1H), 5.64 (d, J 8.0 Hz, 1H), 5.72 (d, J 7.1 Hz, 1H), 7.90 (d, J 7.1 Hz, 1H), 7.98 (dd, J 8.5,
7.3 Hz, 1H), 8.20 (d, 1H, J 7.8 Hz, 1H), 8.31 (d, J 7.8 Hz, 2H), 8.52 (dd, J 8.5, 1.1 Hz, 1H),
8.55 (dd, J 7.3, 1.1 Hz, 1H), 11.28 (s, 1H); δC (150 MHz, DMSO-d6) 162.9, 162.7, 162.7,
150.7, 140.6, 132.5, 131.5, 131.2, 130.9, 129.7, 129.0, 128.7, 128.2, 122.7, 121.9, 101.7, 86.6,
85.9, 68.4, 63.6, 61.1, 47.1, 39.6, 29.4, 27.3, 26.3, 26.2; MS (ESI) [M+H+]+ calcd for
C27H30BrN4O7=601.1298; 603.1277 Found=601.1296; 603.1275; Rf (10% MeOH/CHCl3)
0.13.
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