Synthesis and cytotoxic activity of novel 5-substituted-1-(β-L- arabinofuranosyl) pyrimidine nucleosides

Article abstract of DOI:10.1080/15257770.2012.689410

A series of new 5-halogeno-1-(β-L-arabinofuranosyl)uracils and their cytosine analogues were synthesized by halogenation of ara-L-uridine and ara-L-cytidine, respectively. The 5-(2-thienyl) and 5-halogenothienyl derivatives of both series were also prepared in excellent yields by Stille coupling followed by halogenation. All of these syntheses were based on benzoyl-protected derivatives. In vitro cytotoxicity experiments carried out using L1210 mouse leukemia cells showed that 5-(2-thienyl)- ara-L-uridine was the most potent compound of the new compounds; the majority of the analogues were not effective up to 200 μM concentrations. Copyright Taylor and Francis Group, LLC.

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Synthesis and Cytotoxic Activity of Novel
5-Substituted-1-(β-L-Arabinofuranosyl)
Pyrimidine Nucleosides
Róbert Sendula a , Erika Orbán b , Ferenc Hudecz b , Gyula Sági a
István Jablonkai a
&
a Institute of Organic Chemistry, Research Center of Natural
Sciences, Hungarian Academy of Sciences, Budapest, Hungary
b Research Group of Peptide Chemistry, Hungarian Academy of
Sciences, Eötvös Lóránd University, Budapest, Hungary
To cite this article: Róbert Sendula, Erika Orbán, Ferenc Hudecz, Gyula Sági & István Jablonkai
(2012): Synthesis and Cytotoxic Activity of Novel 5-Substituted-1-(β-L-Arabinofuranosyl) Pyrimidine
Nucleosides, Nucleosides, Nucleotides and Nucleic Acids, 31:6, 482-500
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Nucleosides, Nucleotides and Nucleic Acids, 31:482–500, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.689410
SYNTHESIS AND CYTOTOXIC ACTIVITY OF NOVEL
5-SUBSTITUTED-1-(β-L-ARABINOFURANOSYL)
PYRIMIDINE NUCLEOSIDES
Ro´ bert Sendula,1 Erika Orba´n,2 Ferenc Hudecz,2 Gyula Sa´gi,1
and Istva´n Jablonkai1
1Institute of Organic Chemistry, Research Center of Natural Sciences, Hungarian Academy
of Sciences, Budapest, Hungary
2Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨tvo¨s Lo´ra´nd
University, Budapest, Hungary
A series of new 5-halogeno-1-(ß-L-arabinofuranosyl)uracils and their cytosine analogues were
2
synthesized by halogenation of ara-L-uridine and ara-L-cytidine, respectively. The 5-(2-thienyl) and
5-halogenothienyl derivatives of both series were also prepared in excellent yields by Stille coupling
followed by halogenation. All of these syntheses were based on benzoyl-protected derivatives. In vitro
cytotoxicity experiments carried out using L1210 mouse leukemia cells showed that 5-(2-thienyl)-
ara-L-uridine was the most potent compound of the new compounds; the majority of the analogues
were not effective up to 200 µM concentrations.
Keywords L-nucleosides; 5-substituted pyrimidines; halogenations; cytotoxic activity
INTRODUCTION
Although the first L-nucleoside was synthesized in 1964,[1] relatively
little attention was paid to L-nucleosides due to their unnatural chiral-
ity until the discovery of 2ꢁ-deoxy-3ꢁ-thia-L-cytidine (3TC, Lamivudine).[2]
Lamivudine and analogues were found to be effective against HIV-1, HIV-2,
and HBV viruses at about two orders of magnitude lower concentration
than its D-enantiomer.[3] Combination of 5-fluoro-lamivudine with teno-
fovir disoproxyl fumarate resulted in one of the most effective anti-HIV
cocktails producing substantial and sustained viral supression.[4] Among
Received 20 September 2011; accepted 24 April 2012.
The authors are indebted to Mrs. Erzse´bet Braun for her valuable assistance in the synthetic work
and to Dr. Pal Szabo for the mass spectrometry analyses.
Address correspondence to Dr. Istva´n Jablonkai, Institute of Organic Chemistry, Research Center
of Natural Sciences, Hungarian Academy of Sciences, 1025 Budapest, Pusztaszeri u´t. 59-67, Hungary.
E-mail: jablonkai.istvan@ttk.mta.hu
482

L-arabino-Pyrimidine Nucleosides
483
the 2ꢁ-deoxy-L-nucleosides, L-dT (Telbivudine) and the 3ꢁ-O-valine ester
of L-dC (Valtorcitabine) were found to exhibit extremely high and selec-
tive anti-HBV (but not anti-HIV) activity by strong inhibition of HBV DNA
polymerase.[5] In addition to 2ꢁ,3ꢁ-dideoxy-2ꢁ,3ꢁ-didehydro-L-cytidine deriva-
tives, several fluorinated L-cytidine analogues exhibited marked anti-HIV
and anti-HBV activity.[6,7] Also, several L-nucleosides exhibit antiviral and
anticancer activities.[8–11] These examples demonstrate that unnatural L-
nucleosides with various base- and sugar-modifications have a wide variety
of valuable biological activities irrespective of the number of chiral centers
in the sugar moiety, and have different mechanisms of action. Although the
D-enantiomers of these nucleosides are also effective antiviral and/or antitu-
mor agents, the L-counterparts in most cases have higher metabolic stability
and less toxicity. Therefore, the synthesis and biological evaluation of novel
L-enantiomers of the biologically active D-nucleosides seem to be reasonable.
It is known that the N-glycosidic bond of D-arabino nucleosides is
more resistant to phosphorolytic cleavage than that of the 2ꢁ-deoxy
analogues.[12] The 5-halogeno[13]- and 5-(5-halogenothien-2-yl)[14]-1-(β-D-
arabinofuranosyl)uracil derivatives exhibit comparable effectiveness against
HSV-1, VZV, and VSV as their 2ꢁ-deoxy congeners, whereas the unsubstituted
5-thienyl derivative demonstrated less, but still significant activity.[15]
To investigate the biological activities of several unknown 5-substituted-
pyrimidine β-L-arabinofuranosides, we intended to synthesize new
analogues in both the uracil and cytosine series. More specifically, our
synthetic objectives were to prepare a series of 5-halogeno-1-(β-L-arabino-
furanosyl)uracil/cytosine and 5-(5-halogenothien-2-yl)-1-(β-L-arabino-
furanosyl)uracil/cytosine derivatives with chloro-, bromo-, and iodo-
substitutions to study cytotoxic activities of these new molecules. It should
be noted that even 5-(5-iodo-thien-2-yl)-1-(β-D-arabinofuranosyl)uracil and
cytosine derivatives have not been prepared yet. Prior to the broad-spectrum
antiviral screening, first we intended to investigate the in vitro cytotoxic
effect of these compounds on L1210 mouse leukemia cells.
RESULTS AND DISCUSSION
Chemistry
Our starting material, the ara-L-uridine 1, was synthesized from L-
arabinose in three steps as reported by Holy.[16] Although the further trans-
formations, i.e., halogenations and Stille coupling, can also be performed
directly by using unprotected nucleosides,[13] a recent and detailed com-
parative study[17] suggested that the use of acylated nucleosides as start-
ing materials has been extremely favorable. Based on our earlier studies,
benzoyl-protected derivatives were selected. Indeed, the benzoyl protection

484
R. Sendula et al.
O
N
O
O
N
Cl
Cl
S
NH
NH
NH
O
O
N
O
OH
O
OR
OR
OR
OH
f
OR
b
O
O
OR
7a R = Bz
OH
OR
R = Bz
1
3a
3b
i
i
R = H
R = H
7b
a
O
O
N
O
N
Br
Br
S
NH
NH
NH
N
O
g
O
OR
O
O
OBz
O
OR
c
OR
O
OR
OBz
OR
OR
OBz
2
R = Bz
R = H
8a
8b
4a
4b
R = Bz
i
i
R = H
O
N
O
N
O
N
I
I
S
NH
S
NH
NH
O
OR
O
O
OR
O
O
OR
OR
OR
OR
h
e
d
O
OR
OR
OR
R = Bz
R = H
9a
9b
R = Bz
R = H
R = Bz
R = H
5a
5b
6a
6b
i
i
i
SCHEME 1 Synthesis of 5-substituted-ara-L-uridines. Reagents and conditions: a BzCl, pyridine, 0◦C,
16 hours; b LiCl, CAN, CH3CN:AcOH = 1:1, 80◦C, 8 hours; c LiBr, CAN, CH3CN:AcOH = 3:1, 80◦C,
8 hours; d NIS, TFA, DCE, 60◦C, 3 hours; e 2-(tributylstannyl)-thiophene, Pd(PPh3)4, DMF, 95◦C, 4 hours;
f NCS, pyridine, 100◦C, 5 hours; g Br2, CCl4, DCM, 0◦C, 15 minutes; h NIS, TFA, DCE, 80◦C, 2 hours;
i NaOMe,/MeOH, 4 hours.
proved to be highly advantageous during the product purification and iso-
lation. Benzoylation of 1 afforded the 2ꢁ,3ꢁ,5ꢁ-tri-O-benzoyl nucleoside 2 as
the key intermediate for all target molecules (Scheme 1). Introduction of
chlorine, bromine, and iodine into the C-5 position of pyrimidine nucle-
osides can be performed by various direct halogenation methods.[18] Mild
and efficient halogenation methods for obtaining protected uracil deriva-
tives include the application of N-halosuccinimides and ICl in the presence
of NaN3[13] and lithium halides in the presence of ceric ammonium nitrate
(CAN).[19] The protected 5-chloro- 3a and 5-bromo- 4a derivatives were ob-
tained in nearly 90% yields when LiCl and LiBr were used in chlorination
and bromination reactions in the presence of CAN.[19] Zemplen deacylation
with NaOMe/MeOH offered the required free nucleosides 3b and 4b in
excellent yields. Although iodination of 2 can also be performed applying

L-arabino-Pyrimidine Nucleosides
485
the CAN-mediated method using LiI or I2, the use of N-iodosuccinimide
(NIS) in the presence of TFA in dry dichloroethane resulted in 5a in higher
yield (97%), from which the free 5-iodo-ara-L-uridine 5b was then obtained
by Zemplen deprotection.
The protected iodo derivative 5a also served as starting material for
the preparation of 5-(2-thienyl)-ara-L-uridine 6b. Stille coupling of 5a with
2-tributylstannyl-tiophene in DMF at 95◦C offered the protected 5-(thien-2-
yl) nucleoside 6a in moderate yield (63%). In contrast, thienylation of the
acetyl-protected 5-iodo analogue of the D-series in toluene was reported as a
low yield (33%) reaction.[15] After debenzoylation of 6a, the free 5-(thien-2-
yl)-ara-L-uridine 6b was obtained in good yield (83%). Halogenation of 6a
resulted in various protected 5-(5-halothien-2-yl)-ara-L-uridines (7a, 8a, and
9a) in excellent (90–97%) yields which after deprotection resulted in the
corresponding chloro- 7b, bromo- 8b, and iodo- 9b thienyl-free nucleosides.
Specifically, chlorination of the thienyl moiety was carried out with NCS in
pyridine while the bromo derivative was prepared with Br2 in a mixture of
CCl4 and CH2Cl2. For the iodination, NIS was used in the presence of triflu-
oroacetic acid. In comparision, similar methods for the preparation of 5-(5-
halothien-2-yl)-ara-D-uridines were reported from the acetylated 5-iodo in-
termediates.[14] While the synthesis and biological activity of D-enantiomers
of 7b and 8b have been reported,[14] the 5-(5-iodothien-2-yl)-ara-D-uridine
was not described previously, though its 2ꢁ-deoxy congener is known.[14]
For the transformation of uracil nucleosides to the corresponding cyto-
sine analogues, ammonolysis of the 4-(1,2,4-triazol-1-yl) intermediates is the
most common and reasonable way.[20] 2ꢁ,3ꢁ,5ꢁ-Tri-O-benzoyl-ara-L-cytidine
10a, a common starting material for the synthesis of 5-halogeno-ara-L-
cytidine analogues, was prepared from 2 by ammonolysis of the 4-(1,2,4-
triazol-1-yl) intermediate (Scheme 2). Comparably, ammonolysis and de-
protection of the acetyl-protected analogue yielded 10b in similar overall
yield.[21] However, the use of more stable benzoyl protection as compared
to acetyl is more beneficial in our synthetic strategy since no cleavage of
the benzoyl group occurs during the conversion of uridine to cytosine and
10a can be used for the preparation of all 5-halogeno-pyrimidines. Depro-
tection of 10a afforded the ara-L-cytidine 10b, which was already prepared
but showed no antitumor and anti-HSV activities.[21] Halogenations of 10a
with N-halosuccinimides resulted in new benzoyl-protected 5-chloro- 11a,
5-bromo- 12a, and 5-iodo- 13a ara-L-cytidines in good to excellent yields
(77–94%). After Zemplen debenzoylation, the corresponding free 5-halo
nucleosides 11b, 12b, and 13b were isolated. The D-counterparts of these
compounds such as 5-chloro[22]-, 5-bromo[23]-, and 5-iodo[24]-ara-D-cytidines
have generally been prepared in significantly lower overall yields. The pro-
tected 5-(thien-2-yl)-ara-L-cytidine 14a was synthesized from 6a using the
same procedure that was successfully applied in the transformation of 2 to
10a. Probably due to the steric hindrance of the neighboring thienyl group

486
R. Sendula et al.
NH2
N
NH2
N
Cl
Cl
S
N
O
N
O
b
OR
f
OR
OR
OR
O
O
OR
OR
2
R = Bz
R = H
6a
11a
11b
15a
15b
R = Bz
R = H
i
i
a
e
NH2
N
NH2
NH2
NH2
Br
Br
S
N
S
N
N
N
O
N
O
N
O
N
O
OR
OR
OR
OR
OR
OR
c
g
OR
OR
O
O
O
O
OR
OR
OR
OR
10a
10b
R = Bz
R = H
14a
14b
R = Bz
R = H
12a
12b
R = Bz
R = H
R = Bz
R = H
16a
16b
i
i
i
i
NH2
N
NH2
I
I
S
N
N
O
N
O
OR
OR
d
h
OR
OR
O
O
OR
OR
R = Bz
R = H
17a
17b
R = Bz
R = H
13a
13b
i
i
SCHEME 2 Synthesis of 5-substituted-ara-L-cytidines. Reagents and conditions: a (i) 1,2,4-triazole,
POCl3, pyridine, 0◦C, 16 hours; (ii) cc NH4OH, dioxane, r.t., 8 hours; b NCS, pyridine, 70◦C, 2 hours;
c NBS, pyridine, 50◦C, 4 hours; d NIS, DCE,TFA, 60◦C, 1 hour; e NCS, pyridine, 70◦C, 20 minutes; f Br2,
CCl4, DCM, 0◦C, 20 minutes; g NIS, DCE,TFA, 80◦C, 2 hours; h NaOMe/MeOH, r.t., 4 hours.
in 6a, formation of the 4-triazolyl intermediate was much slower (18 hours
reaction) than in the same conversion with 2 (8 hours reaction). Neverthe-
less, ammonolysis of 4-triazolyl derivative from 6a resulting in the sterically
less crowded 14a required a shorter reaction (4 hours) than in the case
of 10a (8 hours). Halogenations of 14a on the thienyl group were carried
out using NCS, Br2, and NIS followed by Zemplen debenzoylation offered
15b, 16b, and 17b 5-[5-(chloro/bromo/iodo-thien-2-yl]-ara-L-cytidines, re-
spectively. The site of halogenations, i.e., C-5ꢁꢁ-position of the thienyl group,
was confirmed by the vicinal couplings of H3ꢁꢁ and H4ꢁꢁ protons as well as by
the analogy of the same halogenations of 5-(2-thienyl)-ara-D-uridine.[14]
It must be noted that during debenzoylation of cytidine analogues, sig-
nificant amount of the final products remained bound to the resin after
neutralization of the sodium methoxide solution using DOWEX 50 × 2 H-
form resin due to partial protonation of the 4-amino groups. According to

L-arabino-Pyrimidine Nucleosides
487
our earlier experiments,[25] repeated washings with concentrated ammonia
solution were necessary to completely elute the products 10b–17b.
In Vitro Cytotoxicity Studies
The in vitro cytotoxic effect of compounds 3b–17b on L1210 mouse
leukemia cells was characterized by IC50 values as shown in Table 1. In these
measurements, antitumor agents β-D-FdU and 5FU were used as reference
compounds.[26] The ara-L-uridine 1 was slightly cytotoxic, and its 5-chloro-
3b, 5-bromo- 4b, and 5-iodo- 5b analogues were not effective. As a compari-
son, the inhibitory effect of β-L-5FU on the proliferation of murine leukemia
cells (L1210) was also negligible (IC50 294 µM).[26] The ara-L-cytidine 10b,
as well as its 5-chloro- 11b and 5-bromo- 12b derivatives, had no cytotoxic
effect in the concentration range studied, but the 5-iodo compound 13b
exhibited moderate activity. The 5-(2-thienyl)-derivative of ara-L-uridine 6b
(IC50 13.8 µM) was found to be the most active compound, while the same
derivative of ara-L-cytidine 14b was not active at all up to 200 µM concen-
tration. Of the 5-(5-halothien-2-yl)-ara-L-cytidine derivatives, only the 15b
chloro-compound exhibited cytotoxic effect. Among the 5-(5-halothien-2-
yl)-ara-L-uridines, the 7b chloro- and 9b iodo-analogues showed notable
cytotoxicity, while the 8b bromo-compound surprisingly was not effective.
Nevertheless, no structure–activity relationship can be established for
these new molecules. Of the 5-thienyl-ara-L-uridines 6b, 7b, and 9b were
TABLE 1 In vitro cytotoxic effect of the compounds on L1210 mouse leukemia
cells after 72 hours’ incubation time
Compound
IC50 (µM)
s.d.
1
3b
4b
5b
6b
167.5
>200∗
>200
>200
13.8
26.6
2.1
0.3
7b
99.8
8b
9b
>200
47.3
1.6
10b
11b
12b
13b
14b
15b
16b
17b
β-D-FdU
5FU
>200
>200
>200
189.9
>200
37.6
>200
>200
<0.078
1.2
11.3
16.1
0.4
∗The highest concentration tested.

488
R. Sendula et al.
found with IC50 values less than 100 µM. On the other hand, 5-chlorothienyl
analogue 15b showed relatively high cytotoxicity among the generally inac-
tive L-ara-cytidine derivatives. Even if these activities are low as compared to
those of the reference compounds, 6b might be considered as a candidate
for further structural modifications in order to enhance cytotoxicity.
In conclusion, synthesis of several unknown L-nucleosides was accom-
plished with improved yields as reported for D-configured analogues by
applying benzoyl-protected sugar derivatives instead of acetylated interme-
diates. In vitro cytotoxicity experiments carried out with on L1210 mouse
leukemia cells showed that 5-(2-thienyl)-ara-L-uridine (IC50 13.8 µM) was
the most potent compound of the new derivatives while the majority of the
analogues were not effective up to 200 µM concentrations.
EXPERIMENTAL
Chemistry
Melting points were determined with a Boetius apparatus and are uncor-
rected. Infrared spectra were run on Nicolet Avatar 320 FT-IR spectrometer
(Thermo Nicolet, Madison, WI, USA). The NMR spectra were recorded with
1
Varian Gemini-3000 (300 MHz for H and 75 MHz for 13C) spectrometer
(Varian Inc., Palo Alto, CA, USA). Chemical shifts (δ) are given in ppm rela-
tive to Me4Si as internal standard. Electrospray ionization mass spectra (ESI
MS) were recorded on an AB SCIEX API 2000 spectrometer, and high resolu-
tion mass spesctra (HRMS) were determined with a Waters QTOF Premier
instrument. Optical rotatations were measured with a JASCO P-2000 po-
larimeter. Progress of the reactions was monitored by TLC using precoated
aluminum sheets of Kieselgel 60 F254 (Merck). Solvent systems used were as
follows: A) DCM-MeOH, 19:1; B) DCM-MeOH, 9:1; C) DCM-MeOH, 4:1; D)
DCM-MeOH, 1:1; E) EtOAc-hexanes; 6:1, F) EtOAc-hexanes, 1:1; G) EtOAc-
MeOH; 9:1; and H) EtOAc-MeOH,19:1. Spots were detected by UV light at
254 or 360 nm and by charring with 3% ethanolic sulfuric acid followed
by heating. Kieselgel 60 (0.04–0.063 mm, Fluka) was used for column chro-
matography. Methyl propiolate was purchased from Maybridge, cyanamide
from Alfa Aesar, L-arabinose from Reanal, and 2-tributylstannyl-tiophene
from Aldrich, respectively. All (the) other reagents (ceric ammonium ni-
trate (CAN), N-chlorosuccinimide (NCS), N-bromosuccinimide (NBS), N-
iodosuccinimide (NIS)) were bought from Fluka. Pyridine was distilled from
P2O5, dimethylformamide (DMF) from CaH2, dichloromethane (DCM) and
dichloroethane (DCE) from CaCl2 before use.
1-(ß-L-Arabinofuranosyl)-uracil (ara-L-uridine) (1)
The title compound was synthesized from L-arabinose via 2,2ꢁ-anhydro-
L-uridine according to the procedure of Holy´[16] with slight modifications.

L-arabino-Pyrimidine Nucleosides
489
Physical constants and spectral data were identical with those reported in
the literature.[23,25]
2ꢀ,3ꢀ,5ꢀ-Tri-O-benzoyl-ara-L-uridine (2)
To a suspension of 1 (1.80 g, 7.38 mmol) in pyridine (60 mL), benzoyl
chloride (2.74 mL, 23.6 mmol) was added dropwise in an ice-bath with stir-
ring. The mixture was stirred overnight at room temperature, then methanol
(0.5 mL) was added, and stirring was continued for further 0.5 hours. The
mixture was concentrated, then diluted with CH2Cl2 (50 mL), and washed
with saturated aq. NaHCO3 (2 × 30 mL) and finally with water (30 mL).
The organic layer was dried over Na2SO4, filtered, and the solvent was evap-
orated. The product 2 (3.70 g) was obtained as a white foam. Yield 90%;
1
m.p. 178–180◦C; Rf (F) 0.30; H NMR (CDCl3, δ ppm) 4.54 (1H, m, H4ꢁ),
4.87 (2H, m, H5ꢁ), 5.59 (1H, d, H5), 5.65 (1H, t, H2ꢁ), 5.87 (1H, t, H3ꢁ), 6.45
(1H, d, H1ꢁ), 7.26–8.11 (16H, m, Ph + H6), 9.26 (1H, s, NH); 13C
NMR (CDCl3, δ ppm) 63.2 (C5ꢁ), 75.5 (C2ꢁ), 76.6 (C3ꢁ), 81.2 (C4ꢁ), 84.9
(C1ꢁ), 101.8 (C5), 128.5–134.0 (Ph), 140.4 (C6), 149.9 (C2), 162.9 (C4),
164.7–166.2 (PhCO); MS (ESI) m/z [M+H]+ calcd 557.1; found 557.1.
General Procedure for Debenzoylation of the Protected
Nucleosides
The protected nucleoside (1 mmol) was stirred with 0.1 M
NaOMe/MeOH solution (30 mL) for 4 hours at ambient temperature. Wa-
ter (10 mL) was added and the mixture was neutralized with DOWEX 50 ×
2 [H+] resin, which was filtered off and then washed with water (10 mL). In
the case of cytidine analogues, the resin was additionally washed with 25%
aq. ammonia solution (3 × 10 mL). The combined filtrates and washings
were evaporated and the residue was purified by column chromatography on
silica gel using gradient elution (conditions are given for each compound).
5-Chloro-ara-L-uridine (3b)
The mixture of 2 (250 mg, 0.45 mmol), LiCl (23 mg, 0.54 mmol), and
CAN (651 mg, 1.19 mmol) in MeCN-AcOH (1:1, 20 mL) was stirred at
80◦C for 8 hours. The proceeding of the reaction was followed by TLC
(solvent A). The solvent was evaporated and the residue was partitioned
between DCM (50 mL) and water (2 × 20 mL). The organic layer was dried
over Na2SO4, filtered, and concentrated. The crude product was purified
by column chromatography (40 g silica gel, DCM → DCM-MeOH 9:1) to
obtain 3a (230 mg) as a white powder. Yield: 87%; m.p. 136–138◦C; Rf (A)
0.48; MS (ESI) m/z [M+H]+ calcd 591.1; found 591.3.
Deprotection of 3a (230 mg, 0.39 mmol) followed by purification by col-
umn chromatography (20 g of silica gel, DCM → DCM-MeOH, 4:1) yielded

490
R. Sendula et al.
3b (88 mg). Yield 81%; m.p. 227–230◦C; Rf (C) 0.47; [α]25 −72◦ (c 0.25;
D
MeOH); IR (KBr, γ cm−1) 3367 (OH), 3291 (NH), 1718 (C=O, lactam),
1621, 1435 (C=C, ring skeleton pyrimidine moiety), 1282, 1118 (C-O-C),
1
1087, 1046 (Ar-Cl), 1031, 804, 690; H NMR (DMSO-d6, δ ppm) 3.59 (2H,
m, H5ꢁ), 3.72 (1H, m, H4ꢁ), 3.89 (1H, m, H2ꢁ), 3.99 (1H, m, H3ꢁ), 5.14 (1H,
bt, J = 4.8 Hz, 5ꢁOH.), 5.47 (1H, d, J = 3.9 Hz, OH), 5.61 (1H, d, J = 5.1 Hz,
OH), 5.94 (1H, d, J = 4.5 Hz, H1ꢁ), 7.97 (1H, s, H6), 11.78 (1H, bs, NH); 13C
NMR (DMSO-d6, δ ppm) 60.9 (C5ꢁ), 75.5 (C2ꢁ), 75.9 (C3ꢁ), 85.4 (C4ꢁ), 86.0
(C1ꢁ), 106.4 (C5), 139.8 (C6), 150.2 (C2), 159.8 (C4); MS (ESI) m/z [M-H]−
calcd 277.0; found 276.7; HRMS (ESI) m/z [M+H]+ calcd 279.0384; found
279.0384.
5-Bromo-ara-L-uridine (4b)
The mixture of 2 (250 mg, 0.45 mmol), LiBr (47 mg, 0.54 mmol), and
CAN (651 mg, 1.19 mmol) was dissolved in MeCN-AcOH (3:1, 20 mL) and
was stirred at 80◦C for 8 hours. After evaporation, the residue was dissolved
in DCM (50 mL) and washed with water (2 × 20 mL). The organic phase
was dried over Na2SO4 and evaporated. The crude product was purified by
column chromatography on silica gel (40 g, solvent: DCM → DCM-MeOH,
9:1) to obtain 4a (250 mg) as a white solid. Yield 88%; m.p. 142–144◦C;
Rf (A) 0.52; MS (ESI) m/z [M+H]+ calcd 635.1, 637.1; found 635.5,
637.5.
Deprotection of 4a (250 mg, 0.39 mmol) followed by chromatographic
purification (20 g of silica gel, DCM → DCM-MeOH, 4:1) offered 4b
(107 mg). Yield 85%; m.p. 199–201◦C; Rf (C) 0.53; [α]25 −50◦ (c 0.25,
D
MeOH). IR (KBr, γ cm−1) 3380 (OH), 1722 (C O, lactam), 1670 (C O),
1431 (C C, ring skeleton pyrimidine moiety), 1282, 1118 (C-O-C), 1083,
1
1030 (Ar-Br), 804. H NMR (DMSO-d6, δ ppm) 3.59 (2H, m, H5ꢁ), 3.71
(1H, m, H4ꢁ), 3.89 (1H, m, H2ꢁ), 4.00 (1H, m, H3ꢁ), 5.14 (1H, t, J = 5.4 Hz,
5ꢁOH), 5.44 (1H, d, J = 4.5 Hz, OH), 5.60 (1H, d, J = 5.1 Hz, OH), 5.94
(1H, d, J = 4.5 Hz, H1ꢁ), 8.05 (1H, s, H6), 11.77 (1H, bs, NH); 13C NMR
(DMSO-d6, δ ppm) 60.9 (C5ꢁ), 75.5 (C2ꢁ), 75.9 (C3ꢁ), 85.3 (C4ꢁ), 86.0 (C1ꢁ),
94.9 (C5), 142.3 (C6), 150.4 (C2), 159.9 (C4); MS (ESI) m/z [M-H]− calcd
321.0, 323.0; found 320.9, 322.9; HRMS (ESI) m/z [M+H]+ calcd 322.9879;
found 322.9878.
5-Iodo-ara-L-uridine (5b)
To a solution of 2 (1.50 g, 2.70 mmol) and N-iodosuccinimide (675 mg,
3.00 mmol) in DCE (100 mL), trifluoroacetic acid (2.5 mL) was added and
the reaction mixture was stirred for 3 hours at 60◦C. After cooling to ambient
temperature, the solution was extracted with water (2 × 10 mL). The organic
layer was dried over Na2SO4, filtered, and evaporated to dryness. The crude

L-arabino-Pyrimidine Nucleosides
491
product was purified on silica gel to obtain 5a (1.78 g) as a white powder.
Yield 97%; m.p. 209–211◦C; Rf (F) 0.49; MS (ESI) m/z [M+H]+ calcd 683.0;
found 683.0.
Debenzoylation of 5a (660 mg, 0.97 mmol) followed by purification
(20 g of silica gel, DCM → DCM-MeOH, 4:1) offered 5b (320 mg). Yield
89%; m.p. 214–216◦C; Rf (C) 0.54; [α]25 −29◦ (c 0.25, MeOH); IR (KBr,
D
γ cm−1) 3431 (OH), 1662 (C O), 1456 (C C, ring skeleton pyrimidine
1
moiety), 1372, 1279, 1123 (C-O-C), 1063 (Ar-I), 1035, 1020, 617; H NMR
(DMSO-d6, δ ppm) 3.59 (2H, m, H5ꢁ), 3.71 (1H, m, H4ꢁ), 3.88 (1H, m, H2ꢁ),
3.98 (1H, m, H3ꢁ), 5.14 (1H, t, J = 4.8 Hz, 5ꢁOH,), 5.46 (1H, d, J = 4.2 Hz,
OH), 5.60 (1H, d, J = 5.1 Hz, OH), 5.93 (1H, d, J = 4.5 Hz, H1ꢁ), 8.07 (1H,
s, H6), 11.55 (1H, bs, NH); 13C NMR (DMSO-d6, δ ppm) 60.9 (C5ꢁ), 68.2
(C5), 75.5 (C2ꢁ), 75.9 (C3ꢁ), 85.2 (C4ꢁ), 85.8 (C1ꢁ), 147.1 (C6), 150.8 (C2),
161.3 (C4). MS (ESI) m/z [M-H]− calcd 369.0; found 368.6; HRMS (ESI)
m/z [M+H]+ calcd 370.9740; found 370.9741.
5-(2-Thienyl)-ara-L-uridine (6b)
The mixture of 5a (4.54 g, 6.65 mmol) and Pd(PPh3)4 (540 mg,
0.46 mmol) in dry DMF (150 mL) was stirred for 15 minutes at 95◦C and
then 2-(tributylstannyl)-thiophene (2.2 mL, 7.00 mmol) was added. Because
unreacted 5a was detected after 2 hours at 95◦C by TLC (solvent F), ad-
ditional 2-(tributylstannyl)-thiophene (1.2 mL, 3.70 mmol) and Pd(PPh3)4
(270 mg, 0.23 mmol) were added and the mixture was stirred for further
2 hours. After cooling, the solvent was removed by evaporation and the crude
product was purified by column chromatography (40 g of silica gel, hexanes
→ hexanes-EtOAc, 1:1) to obtain 6a (2.71 g) as a white powder. Yield 63%;
m.p. 101–103◦C; Rf (F) 0.54; MS (ESI) m/z [M+H]+ calcd 639.1; found
639.0.
Deprotection of 6a (250 mg, 0.39 mmol) followed by chromatographic
purification (20 g of silica gel, DCM → DCM-MeOH, 9:1) resulted in 6b
(110 mg). Yield 86%; m.p. 224–226◦C; Rf (C) 0.61; [α]25 −11◦ (c 0.25,
D
MeOH); IR (KBr, γ cm−1) 3411 (OH), 1693 (C O, lactam), 1473 (C C, ring
skeleton pyrimidine moiety), 1288 (C C, ring skeleton tiophene moiety),
1
1118 (C-O-C), 1066, 1047, 595; H NMR (DMSO-d6, δ ppm) 3.75 (2H, m,
H5ꢁ), 3.83 (1H, m, H4ꢁ), 4.06 (1H, m, H2ꢁ), 4.18 (1H, m, H3ꢁ), 5.36 (1H, m,
5ꢁOH), 5.57 (1H, m, OH), 5.75 (1H, m, OH), 6.16 (1H, d, J = 5.1 Hz, H1ꢁ),
7.11 (1H, dd, J = 2.7 and 4.2 Hz, H4ꢁꢁ), 7.42 (1H, d, J = 2.7 Hz, H3ꢁꢁ), 7.51
(1H, d, J = 4.2 Hz, H5ꢁꢁ), 8.35 (1H, s, H6); 13C NMR (DMSO-d6, δ ppm) 60.4
(C5ꢁ), 74.7 (C2ꢁ), 76.1 (C3ꢁ), 84.6 (C4ꢁ), 85.6 (C1ꢁ), 107.6 (C5), 123.0 (C3ꢁꢁ),
126.1 (C4ꢁꢁ), 127.2 (C5ꢁꢁ), 134.9 (C2ꢁꢁ), 137.8 (C6), 150.1 (C2), 162.0 (C4).
MS (ESI) m/z [M-H]− calcd 325.1; found 325.0; HRMS (ESI) m/z [M+H]+
calcd 327.0651; found 327.0657.

492
R. Sendula et al.
5-(5-Chlorothien-2-yl)-ara-L-uridine (7b)
A solution of 6a (600 mg, 0.94 mmol) and NCS (180 mg, 1.34 mmol)
in pyridine (30 mL) was stirred for 5 hours at 100◦C and then evaporated.
Purification of the residue by column chromatography (40 g of silica gel,
hexanes → hexanes-EtOAc, 2:1) offered 7a (570 mg) as a white powder.
Yield 90%; m.p. 89–91◦C; Rf (F) 0.61; MS (ESI) m/z [M+Na]+ calcd 695.1;
found 695.3.
Debenzoylation of 7a (100 mg, 0.15 mmol) followed by purification
(20 g silica gel, DCM → DCM-MeOH, 9:1) offered 7b (42 mg). Yield 79%;
m.p. 236–238◦C; Rf (C) 0.67; [α]25D −3◦ (c 0.25, MeOH); IR (KBr, γ cm−1)
3420 (OH), 1696 (C O), lactam), 1674 (C O), 1435 (C C, ring skeleton
pyrimidine moiety), 1288 (C C, ring skeleton tiophene moiety), 1119 (C-O-
C), 1071 (Ar-Cl). 1H NMR (DMSO-d6, δ ppm) 3.66 (2H, m, H5ꢁ), 3.76 (1H,
m, H4ꢁ), 4.00 (1H, m, H2ꢁ), 4.11 (1H, m, H3ꢁ), 5.40 (1H, t, J = 4.8 Hz, 5ꢁOH,
D2O exchangeable), 5.53 (1H, d, J = 4.4 Hz, OH, D2O exchangeable), 5.70
(1H, d, J = 5.2 Hz, OH, D2O exchangeable), 6.22 (1H, d, J = 4.8 Hz, H1ꢁ),
6.89 (1H, d, J = 4.0 Hz, H3ꢁꢁ), 7.19 (1H, d, J = 4.0 Hz, H4ꢁꢁ), 8.44 (1H, s,
H6), 11.81 (1H, brs, NH); 1H NMR (CD3OD, δ ppm) 3.78–3.94 (3H, m, H5ꢁ,
H4ꢁ), 4.12 (1H, t, J = 4.0 Hz, H2ꢁ), 4.24 (1H, t, J = 4.4 Hz, H2ꢁ), 6.22 (1H,
d, J = 4.8 Hz, H1ꢁ), 6.89 (1H, d, J = 4.0 Hz, H3ꢁꢁ), 7.19 (1H, d, J = 4.0 Hz,
H4ꢁꢁ), 8.45 (1H, s, H6); 13C NMR (CD3OD, δ ppm) 61.9 (C5ꢁ), 76.6 (C2ꢁ),
77.5 (C3ꢁ), 85.9 (C4ꢁ), 87.4 (C1ꢁ), 108.9 (C5), 122.9 (C3ꢁꢁ), 126.7 (C4ꢁꢁ), 130.6
(C5ꢁꢁ), 134.5 (C2ꢁꢁ), 138.7 (C6), 151.4 (C2), 163.6 (C4); MS (ESI) m/z [M-
H]− calcd 359.0; found 359.0; HRMS (ESI) m/z [M+H]+ calcd 361.0261;
found 361.0262.
5-(5-Bromothien-2-yl)-ara-L-uridine (8b)
To a solution of 6a (500 mg, 0.78 mmol) in DCM (50 mL) bromine
(48 µl, 0.94 mmol) in CCl4 (10 mL) was added dropwise at 0◦C over 10
minutes. After 15 minutes of stirring, the reaction mixture was washed with
satd. NaHCO3 solution (2 × 30 mL), dried, and evaporated. Purification of
the crude product by column chromatography (50 g of silica gel, hexanes →
hexanes-EtOAc, 1:1) offered 8a (520 mg) as a white powder. Yield 92%; m.p.
91–93◦C; Rf (F) 0.62; MS (ESI) m/z [M+H]+ calcd 719.0, 717.0; found 719.4,
717.1.
Deprotection of 8a (170 mg, 0.24 mmol) followed by purification with
column chromatography (20 g of silica gel, DCM → DCM-MeOH, 4:1) af-
forded pure 8b (85 mg). Yield 89%; m.p. 210–211◦C; Rf (C) 0.67; [α]25D −3◦
(c 0.25, MeOH); IR (KBr, γ cm−1) 3401 (OH), 1694 (C O, lactam), 1474
(C C, ring skeleton pyrimidine moiety), 1432, 1288 (C C, ring skeleton
1
tiophene moiety), 1122 (C-O-C), 1058 (Ar-Br), 794. H NMR (DMSO-d6, δ
ppm) 3.69 (2H, m, H5ꢁ), 3.76 (1H, m, H4ꢁ), 3.99 (1H, m, H2ꢁ), 4.12 (1H, m,

L-arabino-Pyrimidine Nucleosides
493
H3ꢁ), 5.36 (1H, t, J = 4.8 Hz, 5ꢁOH), 5.48 (1H, d, J = 4.5 Hz, OH), 5.67 (1H,
d, J = 5.7 Hz, OH), 6.08 (1H, d, J = 5.4 Hz, H1ꢁ), 7.16 (2H, t, J = 3.9 Hz,
H3ꢁꢁ+ H4ꢁꢁ), 8.39 (1H, s, H6), 11.78 (1H, s, NH); 13C NMR (DMSO-d6, δ
ppm) 60.3 (C5ꢁ), 74.6 (C2ꢁ), 76.2 (C3ꢁ), 84.6 (C4ꢁ), 85.7 (C1ꢁ), 107.0 (C5),
111.7 (C5ꢁꢁ), 122.4 (C3ꢁꢁ), 130.0 (C4ꢁꢁ), 136.5 (C2ꢁꢁ), 137.7(C6), 149.9 (C2),
162.1 (C4); MS (ESI) m/z [M+H]+ calcd 405.0, 407.0; found 404.9, 406.9;
HRMS (ESI) m/z [M+H]+ calcd 404.9756; found 404.9755.
5-(5-Iodothien-2-yl)-ara-L-uridine (9b)
To a solution of 6 (200 mg, 0.31 mmol) and NIS (85 mg, 0.37 mmol)
in CH2Cl2 (10 mL), trifluoroacetic acid (0.5 mL) was added. The reaction
mixture was stirred for 2 hours at 70◦C. The cooled solution was extracted
with satd. NaHCO3 solution (2 × 10 mL) and water (2 × 10 mL). The
organic layer was dried over Na2SO4, filtered, and evaporated to dryness.
The crude product was purified by column chromatography as described
for 8a to obtain 9a (230 mg) as a white powder. Yield 97%; m.p. 107–109◦C;
Rf (F) 0.54; MS (ESI) m/z [M+H]+ calcd 765.0; found 765.1.
Debenzoylation of 9a (200 mg, 0.26 mmol) followed by chromatography
(20 g of silica gel, DCM → DCM-MeOH, 9:1) resulted in 9b (60 mg). Yield
50%; m.p. 162–164◦C; Rf (C) 0.73; [α]25 −19◦ (c 0.25, MeOH); IR (KBr,
D
γ cm−1) 3418 (OH), 2927, 1699 (C O, lactam), 1458 (C C, ring skeleton
pyrimidine moiety), 1419, 1280 (C C, ring skeleton tiophene moiety), 1117
1
(C-O-C), 1050 (Ar-I); H NMR (DMSO-d6, δ ppm) 3.59 (2H, m, H5ꢁ), 3.77
(1H, m, H4ꢁ), 3.94 (1H, m, H2ꢁ), 3.99 (1H, m, H3ꢁ), 4.95 (1H, t, J = 5.1 Hz,
5ꢁOH), 5.44 (1H, d, J = 4.2 Hz, OH), 5.59 (1H,ꢁꢁd, J = 4.8 Hz, OH), 6.02
(1H, d, J = 3.9 Hz, H1ꢁ), 7.33 (2H, bs, H3ꢁꢁ+ H4 ), 8.06 (1H, s, H6), 11.72
(1H, s, NH); 13C NMR (DMSO-d6, δ ppm) 60.9 (C5ꢁ), 75.1 (C2ꢁ), 75.6 (C3ꢁ),
79.3 (C5ꢁꢁ), 82.0 (C4ꢁꢁ), 85.3 (C4ꢁ), 85.8 (C1ꢁ), 105.7 (C5), 139.1 (C6), 142.0
(C3ꢁꢁ), 143.3 (C2ꢁꢁ), 149.5 (C2), 161.3 (C4); MS (ESI) m/z [M-H]− calcd 451.0;
found 451.2; HRMS (ESI) m/z [M+H]+ calcd 452.9617; found 452.9615.
ara-L-cytidine (10b)
To a solution of 1,2,4-triazole (3.20 g, 46.00 mmol) in pyridine (50 mL)
was added POCl3 (1.36 mL,14.80 mmol) at ice-bath temperature and the
mixture was stirred for 30 minutes. The precipitating pyridinium salt was
filtered off and the filtrate was added to the solution of 2 (1.00 g, 1.80 mmol)
in pyridine (20 mL). After 8 hours stirring in an ice-bath, the mixture was
evaporated to dryness. The residue was dissolved in DCM (50 mL) and
washed with water (2 × 10 mL). The organic layer was evaporated and the
residue was redissolved in dioxane (160 mL) and 25% aq. ammonia solution
(40 mL) was added. The mixture was stirred at 10–14◦C for 8 hours then
evaporated. The crude product was purified by column chromatography

494
R. Sendula et al.
(40 g of silica gel, DCM → DCM-MeOH, 9:1) and 10a (700 mg) was obtained
as a white solid. Yield 70%; m.p. 109–112◦C; Rf (A) 0.19; MS (ESI) m/z
[M+Na]+ calcd 578.2; found 578.2.
Debenzoylation of 10a (80 mg, 0.14 mmol) and then purification of the
crude product by column chromatography (20 g of silica gel, DCM → DCM-
MeOH, 2:1) afforded 10b (30 mg). Yield 86%; m.p. 213–216◦C; Rf (C) 0.12;
[α]25 −137◦ (c 0.25, MeOH); IR (KBr, γ cm−1) 3441 (OH), 3358 (NH2),
D
1659 (C O), 1581, 1479 (C C, ring skeleton pyrimidine moiety), 1280,
1112 (C-O-C), 1071, 798; 1H NMR (DMSO-d6, δ ppm) 3.56 (2H, m, H5ꢁ), 3.71
(1H, m, H4ꢁ), 3.86 (1H, m, H2ꢁ), 3.92 (1H, m, H3ꢁ), 5.36 (3H, bs, OH), 5.64
(1H, d, J = 7.5 Hz, H5), 6.01 (1H, d, J = 3.9 Hz, H1ꢁ), 7.00 (2H, bd, NH2),
7.55 (1H, d, J = 7.5 Hz, H6); 13C NMR (DMSO-d6, δ ppm) 61.8 (C5ꢁ), 75.4
(C2ꢁ), 77.0 (C3ꢁ), 85.5 (C4ꢁ), 86.5 (C1ꢁ), 93.0 (C5), 143.6 (C6), 155.9 (C2),
166.3 (C4); MS (ESI) m/z [M+H]+ calcd 244.1; found 244.3; HRMS (ESI)
m/z [M+H]+ calcd 244.0933; found 244.0932.
5-Chloro-ara-L-cytidine (11b)
Solution of 10a (300 mg, 0.54 mmol) and NCS (44 mg, 0.11 mmol) in
pyridine (20 mL) was stirred for 2 hours at 70◦C and then was evaporated.
The residue was redissolved in DCM (50 mL) and washed with water (2 ×
20 mL). The organic layer was dried over Na2SO4 and evaporated. The crude
product was purified by column chromatography (40 g of silica gel, hexanes
→ hexanes-EtOAc, 1:1) to obtain 11 (300 mg) as a white powder. Yield 94%;
m.p. 161–164◦C; Rf (A) 0.92; MS (ESI) m/z [M+H]+ calcd 590.1; found
590.2.
Deprotection of 11a (130 mg, 0.22 mmol) followed by column chro-
matography (20 g of silica gel, DCM → DCM-MeOH; 4:1) afforded 11b
(50 mg). Yield 82%; m.p. 128–129◦C; Rf (C) 0.69; [α]25 −124◦ (c 0.25,
D
MeOH); IR (KBr, γ cm−1) 3343 (OH, NH2), 2839, 2677, 2491, 1644 (C O),
1596, 1501, 1434 (C C, ring skeleton pyrimidine moiety), 1331, 1284, 1121
(C-O-C), 1076 (Ar-Cl), 1037; 1H NMR (DMSO-d6, δ ppm) 3.59 (2H, m, H5ꢁ),
3.79 (1H, m, H4ꢁ), 3.91 (1H, m, H2ꢁ), 4.03 (1H, m, H3ꢁ), 5.07 (1H, bs, OH),
5.52 (1H, bs, OH), 5.61 (1H, bs, OH), 6.00 (1H, d, J = 4.2 Hz, H1ꢁ), 7.93
(1H, d, J = 7.5 Hz, H6), 8.42 and 9.33 (2H, 2 × s, NH2); 13C NMR (DMSO-d6,
δ ppm) 61.4 (C5ꢁ), 75.6 (C2ꢁ), 76.1 (C3ꢁ), 86.4 (C4ꢁ), 87.1 (C1ꢁ), 93.2 (C5),
146.3 (C6), 148.8 (C2), 161.7 (C4). MS (ESI) m/z [M+H]+ calcd 278.0;
found 278.0; HRMS (ESI) m/z [M+H]+ calcd 278.0544; found 278.0545.
5-Bromo-ara-L-cytidine (12b)
Solution of 10 (180 mg, 0.32 mmol) and NBS (180 mg, 0.97 mmol) in
pyridine (20 mL) was stirred for 4 hours at 50◦C and then evaporated to
dryness. The residue taken up in DCM (50 mL) was washed with water (3 ×

L-arabino-Pyrimidine Nucleosides
495
20 mL). Drying over Na2SO4 the solvent was removed in vacuo. Purification
of the crude product was by column chromatography (20 g of silica gel, DCM
→ DCM-MeOH, 9:1) and offered 12a (170 mg, 0.27 mmol) as a white solid.
Yield 82%; m.p. 118–120◦C; Rf (A) 0.39; MS (ESI) m/z [M+H]+ calcd 634.1,
636.1; found 634.3, 636.3.
Deprotection of 12a (280 mg, 0.44 mmol) followed by column chro-
matography (20 g of silica gel, DCM → DCM-MeOH, 4:1) resulted in pure
12b (130 mg). Yield 91%; m.p. 207–210◦C; Rf (C) 0.37; [α]25 −108◦ (c
D
1.0, MeOH); IR (KBr, γ cm−1) 3347 (OH, NH2), 1648 (C O), 1601, 1506
(C C, ring skeleton pyrimidine moiety), 1330, 1281, 1121 (C-O-C), 1086,
1054 (Ar-Br); 1H NMR (DMSO-d6, δ ppm) 3.57 (2H, m, H5ꢁ), 3.73 (1H, m,
H4ꢁ), 3.89 (1H, m, H2ꢁ), 3.94 (1H, m, H3ꢁ), 5.15 (1H, t, J = 5.1 Hz, 5ꢁOH),
5.48 (1H, d, J = 3.9 Hz, OH), 5.51 (1H, d, J = 5.4 Hz, OH), 5.98 (1H, d,
J = 3.9 Hz, H1ꢁ), 6.97–7.75 (2H, 2 × bs, NH2), 7.88 (1H, s, H6); 13C NMR
(DMSO-d6, δ ppm) 60.6 (C5ꢁ), 74.7 (C2ꢁ), 75.7 (C3ꢁ), 84.8 (C4ꢁ), 84.8 (C5),
85.8 (C1ꢁ), 143.3 (C6), 153.6 (C2), 161.7 (C4); MS (ESI) m/z [M-H]− calcd
320.0, 322.0; found 319.8, 321.9; HRMS (ESI) m/z [M+H]+ calcd 322.0039;
found 322.0037.
5-Iodo-ara-L-cytidine (13b)
To a solution of 10a (430 mg, 0.77 mmol) in DCE (20 mL) TFA (0.5 mL)
and NIS (261 mg, 1.16 mmol) were added. The mixture was stirred for 1 hour
at 60◦C and then washed with water (2 × 10 mL). The organic phase was dried
over Na2SO4 and the solvent evaporated in vacuo. The residue was purified
by column chromatography (20 g of silica gel, DCM → DCM-MeOH, 9:1) to
obtain 13a (404 mg) as a white powder. Yield 77%; m.p. 118–120◦C; Rf (A)
0.44; MS (ESI) m/z [M+H]+ calcd 682.1; found 682.2.
Deprotection of 13a (400 mg, 0.59 mmol) followed by column chro-
matography (20 g of silica gel, DCM → DCM-MeOH, 4:1) yielded 13b
(190 mg, 0.51 mmol). Yield 86%; m.p. 210–212◦C; Rf (C) 0.38; [α]25D −36.8◦
(c 0.25, MeOH); IR (KBr, γ cm−1) 3431, 3335 (OH, NH2), 1655 (C O),
1632, 1609, 1504, 1481 (C C, ring skeleton pyrimidine moiety), 1293, 1113
(C-O-C), 1073, 1056 (Ar-I), 1022, 653; 1H NMR (DMSO-d6, δ ppm) 3.57 (2H,
m, H5ꢁ), 3.72 (1H, m, H4ꢁ), 3.87 (1H, m, H2ꢁ), 3.94 (1H, m, H3ꢁ), 5.10 (1H,
d, J = 2.1 Hz, OH), 5.40 (1H, m, OH), 5.45 (1H, m, OH), 5.98 (1H, d, H1ꢁ),
6.53–7.72 (2H, bd, NH2), 7.93 (1H, s, H6); 13C NMR (DMSO-d6, δ ppm) 55.1
(C5), 60.7 (C5ꢁ), 74.8 (C2ꢁ), 75.8 (C3ꢁ), 84.8 (C4ꢁ), 85.8 (C1ꢁ), 148.8 (C6),
153.9 (C2), 163.6 (C4); MS (ESI) m/z [M+H]+ calcd 370.0; found 370.1;
HRMS (ESI) m/z [M+H]+ calcd 369.9900; found 369.9901.
5-(2-Thienyl)-ara-L-cytidine (14b)
The procedure described for the preparation of 10a was used during the
synthesis of 14a starting from 6a (1.60 g, 2.51 mmol). The only difference

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was that the reaction mixture was stirred for 4 hours in an ice-bath followed
by an overnight period in the refrigerator. The product 14a (920 mg) was
obtained as a white crystalline material. Yield 57%; m.p. 112–115◦C; Rf (A)
0.45; MS (ESI) m/z [M+H]+ calcd 638.2; found 638.4.
Deprotection of 14a (87 mg, 0.14 mmol) followed by column chromato-
graphic purification (20 g of silica gel, DCM → DCM-MeOH, 4:1) afforded
14b (36 mg). Yield 81%; m.p. 163–165◦C; Rf (C) 0.39; [α]25 −47◦ (c 0.25,
D
MeOH); IR (KBr, γ cm−1) 3442 (OH), 3346 (NH2), 1653 (C O), 1628,
1506, 1471 (C C, ring skeleton pyrimidine moiety), 1102 (C-O-C), 1058; 1H
NMR (DMSO-d6, δ ppm) 3.56 (2H, m, H5ꢁ), 3.73 (1H, m, H4ꢁ), 3.88 (1H, m,
H2ꢁ), 3.97 (1H, m, H3ꢁ), 5.02 (1H, bs, OH), 5.42 (1H, d, J = 3.3 Hz, OH),
5.49 (1H, d, J = 5.7 Hz, OH), 6.06 (1H, d, J = 3.9 Hz, H1ꢁ), 7.08 (1H, dd,
J = 3.6 and 3.8 Hz, H4ꢁꢁ), 7.12 (1H, d, J = 3.6 Hz, H3ꢁꢁ), 7.54 (1H, d, J =
3.9 Hz, H5ꢁꢁ), 7.70 (1H, s, H6), 7.85 (2H, m, NH2); 13C NMR (DMSO-d6, δ
ppm) 61.5 (C5ꢁ), 75.6 (C2ꢁ), 76.7 (C3ꢁ), 85.5 (C4ꢁ), 86.4 (C1ꢁ), 99.9 (C5),
126.7 (C3ꢁꢁ), 127.6 (C4ꢁꢁ), 128.8 (C5ꢁꢁ), 128.9 (C-2ꢁꢁ), 143.3 (C6), 154.7 (C2),
163.7 (C4); MS (ESI) m/z [M+H]+ calcd 326.1; found 326.0; HRMS (ESI)
m/z [M+H]+ calcd 326.0811; found 326.0811.
5-(5-Chlorothien-2-yl)-ara-L-cytidine (15b)
A solution of 14a (350 mg, 0.55 mmol) and NCS (100 mg, 0.75 mmol)
in pyridine (20 mL) was stirred for 20 minutes at 70◦C. The solution was
evaporated to dryness and the residue taken up in DCM (50 mL) was washed
with water (2 × 30 mL). The organic layer dried over Na2SO4 was filtered and
evaporated. Purification of the crude product by column chromatography
(40 g of silica gel, hexanes → hexanes-EtOAc, 1:1) offered 15a (300 mg) as
a white solid. Yield 81%; m.p. 93–95◦C; Rf (A) 0.89; MS (ESI) m/z [M+H]+
calcd 672.1; found 672.1.
Deprotection of 15a (100 mg, 0.15 mmol) and column chromatography
(20 g of silica gel, DCM → DCM-MeOH, 9:1) of the crude product afforded
(42 mg) 15b. Yield 79%, m.p. 115–117◦C; Rf (C) 0.37; [α]25 −40◦ (c 0.25,
D
MeOH); IR (KBr, γ cm−1) 3419 (ON, NH2), 2929, 2360, 1699 (C O), 1636,
1458 (C C, ring skeleton pyrimidine moiety), 1281 (C C, ring skeleton
tiophene moiety), 1121 (C-O-C), 1072 (Ar-Cl); 1H NMR (DMSO-d6, δ ppm)
3.56 (2H, m, H5ꢁ), 3.77 (1H, m, H4ꢁ), 3.89 (1H, m, H2ꢁ), 4.09 (1H, m, H3ꢁ),
5.30–5.85 (2H, bs, 2OH), 5.55 (1H, s, OH), 6.02 (1H, d, J = 4.5 Hz, H1ꢁ),
7.03 (1H, d, J = 3.9 Hz, H4ꢁꢁ), 7.11 (1H, d, J = 3.9 Hz, H3ꢁꢁ), 8.10 (1H, s,
H6), 8.60–9.05 (2H, bd, NH2); 13C NMR (DMSO-d6, δ ppm) 59.9 (C5ꢁ), 74.3
(C2ꢁ), 75.2 (C3ꢁ), 85.0 (C4ꢁ), 86.1 (C1ꢁ), 98.8 (C5), 127.8–129.6 (C3ꢁꢁ, C4ꢁꢁ,
C5ꢁꢁ), 129.8 (C2ꢁꢁ), 145.3 (C6), 147.3 (C2), 159.3 (C4); MS (ESI) m/z [M+H]+
calcd 360.0; found 360.1; HRMS (ESI) m/z [M+H]+ calcd 360.0421; found
360.0420.

L-arabino-Pyrimidine Nucleosides
497
5-(5-Bromothien-2-yl)-ara-L-cytidine (16b)
To a solution of 14a (350 mg, 0.55 mmol) in CHCl3 (20 mL) was added
the solution of Br2 (60 µl, 1.10 mmol) in CCl4 (10 mL) dropwise over
10 minutes at ice-bath temperature. After 15 minutes stirring, the solution
was washed with water (2 × 30 mL). The organic layer dried over Na2SO4
was filtered and evaporated. The crude product was purified by column
chromatography (50 g of silica gel, hexanes → hexanes-EtOAc, 1:1) to give
16a (360 mg) as a yellow powder. Yield 91%; m.p. 120–122◦C; Rf (A) 0.46;
MS (ESI) m/z [M+Na]+ calcd 740.1, 738.1, 7; found 740.3, 738.3.
Deprotection of 16a (170 mg, 0.24 mmol) followed by column chromato-
graphic purification (20 g of silica gel, DCM → DCM-MeOH, 4:1) resulted in
16b (75 mg). Yield 78%; m.p. 163–165◦C; Rf (C) 0.41; [α]25 −26◦ (c 0.25,
D
MeOH); IR (KBr, γ cm−1) 3445 (OH), 3343 (NH2), 1658 (C O), 1629,
1578, 1508, 1473 (C C, ring skeleton pyrimidine moiety), 1291 (C C, ring
skeleton tiophene moiety), 1103 (C-O-C), 1068, 1054 (Ar-Br), 1007; 1H NMR
(DMSO-d6, δ ppm) 3.55 (2H, m, H5ꢁ), 3.73 (1H, m, H4ꢁ), 3.89 (1H, m, H2ꢁ),
3.98 (1H, m, H3ꢁ), 5.05 (1H, t, J = 5.4 Hz, 5ꢁOH, D2O exchangeable), 5.41
(1H, d, J = 4.2 Hz, OH, D2O exchangeable), 5.47 (1H, d, J = 5.7 Hz, OH
D2O exchangeable), 6.05 (1H, d, J = 4.2 Hz, H1ꢁ), 6.50–7.60 (2H, bs, NH2,
D2O exchangeable), 6.90 (1H, d, J = 3.9 Hz, H4ꢁꢁ), 7.21 (1H, d, J = 3.6 Hz,
H3ꢁꢁ), 7.70 (1H, s, H6); 13C NMR (DMSO-d6, δ ppm) 66.1 (C5ꢁ), 80.4 (C2ꢁ),
81.2 (C3ꢁ), 90.2 (C4ꢁ), 91.2 (C1ꢁ), 103.8 (C5), 116.1 (C5ꢁꢁ), 133.7 (C3ꢁꢁ), 136.6
(C4ꢁꢁ), 142.2 (C2ꢁꢁ), 148.5 (C6), 159.5 (C2), 168.5 (C4); MS (ESI) m/z [M-
H]− calcd 402.0, 404.0; found 402.7, 403.7; HRMS (ESI) m/z [M+H]+ calcd
403.9916; found 403.9916.
5-(5-Iodothien-2-yl)-ara-L-cytidine (17b)
To a solution of 14a (300 mg, 0.47 mmol) dissolved in DCE (20 mL) TFA
(0.5 mL) and NIS (127 mg, 0.56 mmol) were added. The mixture was stirred
for 2 hours at 80◦C then washed with water (2 × 10 mL). The organic layer
dried over Na2SO4 was evaporated and the residue was purified by column
chromatography (40 g of silica gel, EtOAc) to give 17a (220 mg) as a white
solid. Yield 61%; m.p. 133–135◦C, Rf (H) 0.58; MS (ESI) m/z [M+H]+ calcd
764.1; found 764.2.
Deprotection of 17a (393 mg, 0.51 mmol) followed by column chro-
matography (20 g of silica gel, DCM → DCM-MeOH, 9:1) offered 17b
(90 mg). Yield 39%; m.p. 199–201◦C; Rf (C) 0.45; [α]25 −11◦ (c 0.25,
D
MeOH); IR (KBr, γ cm−1) 3447 (OH), 3347 (NH2), 2933, 1656 (C O),
1633, 1578, 1472 (C C, ring skeleton pyrimidine moiety), 1290 (C C, ring
1
skeleton tiophenene moiety), 1102 (C-O-C), 1067 (Ar-I), 1053; H NMR
(DMSO-d6, δ ppm) 3.55 (2H, m, H5ꢁ), 3.71 (1H, m, H4ꢁ), 3.90 (1H, m, H2ꢁ),
3.98 (1H, m, H3ꢁ), 5.00–5.80 (3H, bs, 3OH D2O exchangeable), 6.05 (1H,

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d, J = 4.2 Hz, H1ꢁ), 6.40–7.50 (2H, bs, NH2 D2O exchangeable), 6.78 (1H,
d, J = 3.6 Hz, H4ꢁꢁ), 7.30 (1H, d, J = 3.6 Hz, H3ꢁꢁ), 7.68 (1H, s, H6); 13C
NMR (DMSO-d6, δ ppm) 60.7 (C5ꢁ), 67.4 (C5ꢁꢁ), 75.0 (C2ꢁ), 75.7 (C3ꢁ), 84.6
(C4ꢁ), 85.6 (C1ꢁ), 98.5 (C5), 127.0 (C3ꢁꢁ), 129.0 (C4ꢁꢁ), 137.6 (C2ꢁꢁ), 140.7
(C6), 154.1 (C2), 162.9 (C4); MS (ESI) m/z [M+H]+ calcd 452.0; found
452.1; HRMS (ESI) m/z [M+Na]+ calcd 473.9597; found 473.9594.
Determination of In Vitro Cytotoxic Effect of the Compounds
L1210 mouse lymphotic leukemia cells (ATCC: CCL-219) were cultured
in RPMI-1640 medium supplemented with 10% FCS (fetal calf serum, Sigma
Ltd.), L-glutamine (2 mM), and gentamicin (160 µg/mL). Cell culture was
maintained at 37◦C in a humidified atmosphere with 5% CO2. The in vitro cy-
totoxic effect of the compounds was evaluated by the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide-assay (MTT-assay).[27] For the exper-
iment, 5 × 103 cells per well were plated on 96-well plates. After 24 hours
incubation at 37◦C, cells were treated for 72 hours with the compounds dis-
solved in 0.5% DMSO containing serum-free medium. The compounds were
tested in the 0.078–200 µM concentration range. Cells treated with serum-
free medium for 72 hours were used as negative control. After the treatment,
cells were washed with serum-free medium and MTT-assay was carried out.
MTT solution was added to each well (final concentration: 367 µg/mL).
After 3.5 hours incubation, purple crystals were formed by mitochondrial
dehydrogenase enzyme of living cells. Cells were centrifuged for 5 minutes
at 863 g and the supernatant was removed. Crystals were dissolved in DMSO
and the optical density (OD) of the samples was measured at λ = 540 and
620 nm using an ELISA Reader (Labsystems MS reader, Finland). OD620 was
substracted from OD540. The percentage of cytotoxicity was calculated using
the following equation:
Cytotoxicity% = [1 − (ODtreated/ODcontrol)] × 100,
where ODtreated and ODcontrol correspond to the optical densities of treated
and control cells, respectively. Cytotoxicity percentage was plotted as a func-
tion of concentration, fitted to a sigmoidal curve, and the IC50 value was
determined on the basis of this curve.
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