Bicyclic cytarabine analogues: Synthesis and investigation of antitumor properties of novel, 6-aryl- and 6-alkyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one arabinosides

Article abstract of DOI:10.1016/j.tet.2015.09.015

A series of sixteen hitherto unknown Cytarabine analogues bearing a bicyclic 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one base modified with aryl, pyridyl, benzyl and alkyl substituents was prepared in a straightforward approach. This is the one of the few exampl

Full text of DOI:10.1016/j.tet.2015.09.015

Accepted Manuscript
Bicyclic Cytarabine Analogues: Synthesis and Investigation of Antitumor Properties of
Novel, 6-Aryl- and 6-Alkyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one Arabinosides
Adam Mieczkowski, Malgorzata Makowska, Justyna Sekula, Ewelina Tomczyk, Ewa
Zalewska, Anna Nasulewicz-Goldeman, Joanna Wietrzyk
PII:
S0040-4020(15)30045-4
10.1016/j.tet.2015.09.015
TET 27112
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 22 January 2015
Revised Date: 13 August 2015
Accepted Date: 4 September 2015
Please cite this article as: Mieczkowski A, Makowska M, Sekula J, Tomczyk E, Zalewska E, Nasulewicz-
Goldeman A, Wietrzyk J, Bicyclic Cytarabine Analogues: Synthesis and Investigation of Antitumor
Properties of Novel, 6-Aryl- and 6-Alkyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one Arabinosides, Tetrahedron
(2015), doi: 10.1016/j.tet.2015.09.015.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT:
R
H
O
NH2
N
NH
N
N
N
O
N
O
O
N
O
O
5-steps
O
HO
HO
OH
HO
OH
OH
HO
HO
AraC
HO
bicyclic Cytarabine
analogues
araU
R = Ph, substituted Ph, 2-pyridyl,
benzyl, alkyl

ACCEPTED MANUSCRIPT
Bicyclic Cytarabine Analogues: Synthesis and Investigation of Antitumor
Properties of Novel, 6-Aryl- and 6-Alkyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one
Arabinosides
Adam Mieczkowski,1* Malgorzata Makowska, 1 Justyna Sekula,1 Ewelina Tomczyk,1
Ewa Zalewska,1 Anna Nasulewicz-Goldeman,2 Joanna Wietrzyk2
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences 5a, Pawinskiego Street, 02-106
Warsaw, Poland
2Institute of Immunology and Experimental Therapy, Polish Academy of Sciences 12, R. Weigl Street,
53-114 Wroclaw, Poland
Key words: bicyclic pyrimidine nucleoside analogues (BCNAs), arabinonucleosides,
Cytarabine analogues, cytotoxicity
* Corresponding author. Address: Institute of Biochemistry and Biophysics, Polish
Academy of Sciences, 5A Pawinskiego St., 02-106 Warszawa, Poland. Fax +48
22 592 21 90, E-mail address: amiecz@ibb.waw.pl
1

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ABSTRACT:
A series of sixteen hitherto unknown Cytarabine analogues bearing a bicyclic 3H-
pyrrolo[2,3-d]pyrimidin-2(7H)-one base modified with aryl, pyridyl, benzyl and alkyl
substituents was prepared in a straightforward approach. This is the one of the few
examples of the synthesis of pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides and the
first example of pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides possessing arabinose
moiety. For the first time, the conversion of the furopyrimidine arabinoside products
to a series of novel pyrrolopyrimidines by ammonolysis reaction was thoroughly
investigated using aqueous and methanolic reaction conditions under classical and
micro-wave heating. This approach resulted in a small library of compounds, which
were evaluated for their antiproliferative properties against HL-60 and Jurkat E6.1
cell lines. All synthesised compounds exhibited a weaker cytotoxic effect in
comparision to the mother compound. Of all the tested compounds, the derivative
bearing a 4-n-pentylphenyl substituent exhibited the highest antiproliferative activity.
2

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1. Introduction
Cytarabine ((1-β-
D
-arabinofuranosyl)cytosine), cytosine arabinoside, Ara-C, 1,
Fig. 1) is an effective drug widely used for the treatment of acute myelogenous
leukemia and a lymphocytic leukemia.1 Early experiments revealed a wide range of
anticancer activity against numerous cell lines of sarcomas (Nakahara-Fukuoka
sarcoma, reticulum cell sarcoma, ascites sarcoma-180), adenocarcinomas
(adenocarcinoma-755, spontaneous mammary adenocarcinoma), and Ehrlich ascites
carcinoma.2-4 Cytarabine also exhibited remarkable synergistic effect with other
anticancer drugs like Daunorubicin, Vinblastin and cis-diaminodichloroplatin (II)
(CDDP).5,6 However, the main disadvantages of antitumor therapy with Cytarabine
are related to its acute and chronic toxicity to internal organs7-12 and its poor
bioavailability; it is quickly metabolized by cytidine deaminase to
a
pharmacologically inactive metabolite, uracil arabinoside (AraU). For this reason, any
new approaches leading to improvement of the pharmacokinetics of Cytarabine are
highly desirable and many efforts have been directed toward the discovery of new,
cytidine deaminase-resistant Cytarabine analogues. It’s highly desiderable, that they
retain retain the antitumor activity exhibiting lower toxicity and better bioavailability
then the mother compound. Such analogues include, but are not limited to,
Enocytabine (2, N4-behenoyl-1-(β-
D-arabinofuranosyl)cytosine, BH-AC), which bears
a highly lipophilic group at the 4-amino position of the cytosine moiety of Cytarabine
(1)13,14 and Ancitabine (3), a cyclonucleoside analogue possessing an additional
covalent O2’,2-linkage15,16 in the structure. Krolikiewicz et al reported the synthesis of
tetrahydro-2H-pyrrolo[2,3-d]pyrimidine analogue of Cytarabine 4,17 but this
derivative was deprived of any biological activity. All decribed structures are
represented in the Figure 1.
3

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O
NH2
NH
N
HN
NH(CH2)20CH3
N
N
O
N
N
O
O
O
N
O
HO
HO
O
OH
HO
OH
HO
HO
HO
1
2
3
R
H
H
N
N
N
N
N
O
N
O
O
O
HO
HO
OH
OH
HO
HO
4
5
Fig.1 Cytarabine (1) and its derivatives 2-5
Nucleosides possessing bicyclic bases are known as efficient cytidine
mimetics, forming stable Watson-Crick pairs with guanidine in the DNA and RNA
helices.18,19 Bicyclic pyrimidine nucleoside analogues (BCNAs) are widely known for
their antiviral activity against various RNA and DNA viruses such as: VZV,20,21
HCMV,22,23 HCV,24,25 HBV26 and the vaccinia virus (VACV).27 They have not been,
however, investigated as possible anticancer agents yet. BCNAs possessing an
arabinofuranosyl ring and furo[2,3-d]pyrimidin-2(3H)-one as base were reported as
potential antivirial compounds, but exhibited weak activity against VZV and HCMV
strains.28 Gazivoda reported that 4′,5′-didehydro-
L-ascorbic acid derivatives
possessing bicyclic furo[2,3-d]pyrimidine bases exhibit pronounced cytostatic activity
against malignant leukemia (L1210).29 However, earlier reports revealed that bicyclic
nucleosides possessing either oxazolo[5,4-d]pyrimidine30 or thieno[5,4-d]pyrimidine31
bases exhibit remarkable in vivo and in vitro antileucemic activity against L1210 cells,
but there is very little additional literature data in that subject.
4

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In our ongoing quest for the design and synthesis of new medicinally relevant
heterocyclic derivatives32-36 and compounds exhibiting antitumor activity,37-40 we
envisioned the possibility of synthesising novel, cytidine deaminase-resistant,
Cytarabine analogues bearing a bicyclic, pyrrolo[2,3-d]pyrimidin-2(7H)-one base at
the arabinose moiety (Fig. 1, 5). There are only a few examples in the literature
concerning the pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides, and no examples of
such compounds possessing arabinose moiety. We assumed, that designed
pyrrolo[2,3-d]pyrimidin-2(7H)-one arabinosides could serve as covenient analogues
of Cytarabine, possibly retaining the anticancer activity resulting from undergoing
phosphorylation to appropriate triphosphates by cellural enzymes. Such triphosphates
can inhibit DNA polymerases and terminate DNA chain elongantion. Addtionally, as
an amine group is a part of pyrrole ring, these analogues would not serve as substrates
for the cytidine deaminase. This in consequence, would improve bioavailability and
extend the half-life of the compounds. We also assumed that the addition of lipophilic
substituents to the pyrrole ring would increase the penetration of such compounds
through the blood brain-barrier in reference to Cytarabine (1).
2. Results and Discussion
The synthesis of designed compounds was conducted starting from
commercially available 1-(β-D-arabinofuranosyl)uracil (6) protected by acetyl groups.
Acetylation reaction was performed with acetic anhydride in the presence of
triethylamine and a catalytic amount of DMAP (Scheme 1).41 After crystallization
from ethanol, the triacetate was subjected to the iodination reaction using I2 in the
presence of cerium ammonium nitrate,42 leading to formation of 1-(2,3,5-tri-O-acetyl-
β-D-arabinofuranosyl)-5-iodouracil (7). Intermediate product 7 was then applied in a
series of Sonogashira reactions using 16 alkynes, under Pd(PPh3)4/CuI catalysis [26-
27], which resulted in formation of 5-alkynyl derivatives 8 as the main products and
bicyclic furanopyrimidines 9 as minor ones.43 For complete cycloisomerisation,
obtained mixtures of compounds 8 and 9 were heated for additional 12 h in 50-60 °C
in the presence of one more equivalent of copper iodide.
5

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O
N
O
R
O
N
I
NH
NH
NH
a,b
c
O
N
O
O
O
O
O
HO
AcO
AcO
OH
OAc
OAc
HO
AcO
AcO
6
7
8a-p
d
R
R
R
H
N
O
O
N
N
N
e
N
O
N
O
N
O
O
O
O
HO
HO
AcO
OH
OH
OAc
HO
HO
AcO
5a-p (24-94%)
10a-p
9a-p (38-74%)
Cl
Cl
N
Cl
Cl
R =
a
b
c
d
e
F
HO
F
CH3(CH2)4
F3CO
F
f
g
h
i
j
O
NC
H3C
HO
7
9
k
l
m
n
o
p
Scheme 1. Reagents and conditions: a) Ac2O, TEA, DMAP, MeCN, 20 h, rt b)
(NH4)2Ce(NO3)6, I2, MeCN, 3 h, 80 °C c) R-C≡CH, Pd(PPh3)4, CuI, TEA, DMF, 24h,
rt d) CuI, TEA, DMF, 12 h, 50-60 °C e) 7N methanolic ammonia, 60 °C, 24-48 h, mv
or 25% NH4OH, rt, 1-4 d.
According to the literature data, transformation of the furo[2,3-d]pyrimidin-
2(3H)-one nucleosides to the 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides
6

ACCEPTED MANUSCRIPT
could be performed with aqueous or methanolic solutions of ammonia, but
comperative studies of both methods were not reported. For this reason, we then
tested both reagents for the last step of the synthesis; the removal the acetyl groups
and concomitant conversion of the furane ring to the pyrrole ring. Treatment of
compound 9 with 7N methanolic ammonia at room temperature led to the fast
removal of the acetyl groups followed by rather slow conversion of the deprotected
furo[2,3-d]pyrimidin-2(3H)-one nucleosides 10 to the 3H-pyrrolo[2,3-d]pyrimidin-
2(7H)-one nucleosides 5.33,44 We found that almost complete conversion of furane to
the pyrrole ring could be performed under microwave heating at 60 °C. When the
time reaction is extendeed to 24-48 h, the final compounds could be obtained with
moderate to high yields (32-94%). Application of aqueous solution of ammonia
slightly accelerated the transformation of the furane into the pyrrole ring, however it
also led to formation of by-products, which in some cases were difficult to separate
from the main product. When the reaction temperature was raised to 60 °C, the
number of by-products increased, therefore most reactions with NH4OH were carried
out at room temperature with prolonged reaction time (up to the 4 days in case of
products 5o and 5p bearing long liphophilic chains, which limited the solubility in the
aqueous solutions).
All new Cytarabine analogues were tested for their anti-proliferative activity
against HL-60 (human promyelocytic leukemia) and Jurkat E6.1 (human acute T cell
leukemia) cells. Compound 5g showed the highest anti-proliferative activity with IC50
values 68 and 61 µM for HL-60 and Jurkat E6.1 cells, respectively. Compounds 5a,
5c-5f and 5h-5j showed rather poor anti-proliferative properties with IC50 values
ranging from 95 to 203 µM. Compounds 5b, 5k-5n and 5p did not influence the
proliferation rate of HL-60 and Jurkat E6.1 cells. The reference IC50 values calculated
for Cytarabine were 0.185 and 0.018 µM for HL-60 and Jurkat E6.1 cells,
respectively. The significantly lower anti-proliferative properties of the synthesised
compounds could be attributed to their different behaviour in the cell. It’s likely, that
they could undergo intracellular coversion to appropiate triphosphates, but would not
be used as substrates in the DNA and RNA synthesis reactions. Therefore, they could
not serve as agents stopping the synthesis of nucleic acids in the cell.
3. Conclusion
7

ACCEPTED MANUSCRIPT
We have efficiently synthesised a series of sixteen hitherto unknown Cytarabine
analogues bearing a bicyclic 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one base modified
with aryl, pyridyl, benzyl and alkyl substituents. This is the first example of synthesis
of 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides possessing arabinose moiety.
The key intermediate product, acetylated 5-iodo-3-(β-D-arabinofuranosyl) uracil, was
applied to Sonogashira couplings with appropiate terminal alkynes. The Sonogashira
reaction products were usually accompanied by furo[2,3-d]pyrimidin-2(3H)-one by-
products resulting from the metal-catalyzed cycloisomerization reaction, and were
directly applied to the cycloisomerization step in the presence of copper(I) iodide. In
the final step, the obtained furo[2,3-d]pyrimidin-2(3H)-one arabinosides were treated
with the aqueous or methanolic ammonia, which resulted in the deprotection of the
hydroxyl groups and transformation of furane ring into the pyrrole ring. A brief
screening of amonolysis reaction conditions, using aqueous versus methanolic
ammonia solutions and traditional versus microwave heating revealed, that optimal
conditions for this transformation could be achived using metanolic ammonia under
the microwave heating. The resulting library of compounds was evaluated for their
anti-proliferative effect against HL-60 human promyelocytic leukemia cells and
Jurkat E6.1 human acute T cell leukemia cells. Compound 5g, bearing the 4-n-
pentylphenyl substituent attached to the pyrrole ring, showed the highest anti-
proliferative activity with IC50 values 68 and 61 µM for HL-60 and Jurkat E6.1
respctively. Although the synthetised compounds exhibited a rather weak cytotoxic
effect on the investigated cell lines, the results and observed effects could help in the
design of more active and selective inhibitors.
4. Experimental section
4.1. Chemistry
Commercially available chemicals were of reagent grade purity and used as
received. The reactions were monitored by thin layer chromatography (TLC) using
silica gel plates (Kieselgel 60F254, E. Merck). Column chromatography was
19
performed on Silica Gel 60M (0.040-0.063 mm, E. Merck). The 1H, 13C NMR and
F spectra were recorded on Varian Unity Plus spectrometer (500 MHz) in MeOH-d4,
CDCl3 and DMSO-d6. Chemical shift values are reported in parts per million relative
to SiMe4 as internal reference. Fluorine 19 F spectra were measured using CFCl3 as an
internal reference. The resonance assignments are based on peak integration, peak
8

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multiplicity and 2D correlation experiments. Multiplets were assigned as s (singlet), d
(doublet), t (triplet), q (quartet), dd (doublet of doublet), dt (doublet of triplet), ddd
(doublet of doublet of doublet), m (multiplet), and bs (broad singlet). HRMS spectra
were performed on LTQ Orbitrap Velos Thermo Scientific. The microwave-assisted
reactions were performed in the CEM Discover SP microwave reactor, set to 80 Watt
input power at 60-65 °C, in 35 mL glass tubes.
The synthesis of 1-(2,3,5-tri-O-acetyl-β-
D
-arabinofuranosyl)-5-iodouracil (7)
-arabinofuranosyl)uracil (6) was
5.00 g (20.49 mmol, 1 eq.) of 1-(β-
D
dispersed in the 40 mL of dry MeCN. Then 25 mg (0.20 mmol, 0.01 eq.) of DMAP,
11.55 mL (84.00 mmol, 4.1 eq.) of triethylamine and 7.80 mL (81.96 mmol, 4 eq.) of
acetic anhydride were added. Reaction mixture was stirred at room temperature for 20
h. After this time all volatiles were evaporated under the reduced pressure. The
residue was dissolved in CHCl3 and washed twice with water. The combined organic
layers were dried over magnesium sulphate and the solvent was removed on an
evaporator. After crystallization from ethanol, 6.575 g (17.76 mmol, 87% yield) of 1-
(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)uracil were obtained. 3.50 g Of obtained
(9.45 mmol, 1eq.) triacetate were dissolved in 150 mL of MeCN, then 2.59 g (4.72
mmol, 0.50 eq.) of ammonium cerium(VI) nitrate and 1.43 g (5.63 mmol, 0.6 eq.) of
iodine was added. Obtained suspension was stirred in 80°C for 3 h, and then solvent
was removed by evaporation under reduced pressure. The residue was dissolved in
EtOAc and washed with brine, sodium thiosulfate solution and water. Combined
organic phases were dried over MgSO4. Analytically pure compound was obtained by
crystallization from ethanol. Yield: 4.49 g (75%). Mp: 167.7-168.7 °C (lit. 168 °C). 1H
NMR (500 MHz, CDCl3): 9.24 (s, 1H, NH), 7.92 (s, 1H, H6), 6.31 (d, J = 4.0 Hz, 1H,
H1’), 5.41 (dd, J1 = 2.0 Hz, J2 = 4.0 Hz, 1H, H2’), 5.13 (q, J = 2.0 Hz, 1H, H3’), 4.46
(dd, J1 = 6.0 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.42 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a),
4.25-4.19 (m, 1H, H4’), 2.19 (s, 3H, MeAc), 2.16 (s, 3H, MeAc), 2.07 (s, 3H, MeAc) 13C
NMR (125 MHz, CDCl3): 170.5, 169.6, 168.5, 159.5, 149.5, 145.0, 84.3, 80.7, 76.2,
74.4, 67.5, 62.5, 20.9, 20.7, 20.4. HRMS (ESI): m/z [M+H]+ calcd for C15H17IN2O9:
497.00515, found: 497.00439.
9

ACCEPTED MANUSCRIPT
General procedure for the synthesis of protected 6-substituted furo[2,3-
d]pyrimidin-2(3H)-one arabinosides (9)
1-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-5-iodouracil (7) (1 eq.) was dissolved in
anhydrous DMF (5 mL by 1 mmol of nucleoside) under Ar atmosphere. To the
obtained solution, the appropriate terminal alkine (3 eq.), triethylamine (1.9 eq.),
copper iodide (0.2 eq.), and tetrakis(triphenylphosphine)palladium(0) (0.1 eq.) were
added and reaction mixture was stirred at room temperature for 24 h. On the next day,
the dark solution was heated to the 50-60 °C, then additional amount of copper iodide
(1.0 eq) was added and stirring was continued for additional 12 hrs. Reaction mixture
was diluted with ethyl acetate (v/v DMF:EtOAc 1:4), the organic phase was washed
three times with water (v/v DMF:H2O 1:4) and dried over magnesium sulphate.
Volatiles were evaporated under reduced pressure and the oily residue was
chromatographed using an appropriate eluent. Analytically pure compounds were
obtained by crystallization from methanol or acetone.
3-(2,3,5-Tri-O-acetyl-β-
D
-arabinofuranosyl)-6-phenylfuro[2,3-d]pyrimidin-2(3H)-
20
one (9a). Yield: 38%, white crystals, mp.: 219.1-219.8 °C. [α]D +89.9 (c 1.0,
DMSO); 1H NMR (500 MHz, CDCl3): 8.30 (s, 1H, H4), 7.81-7.75 (m, 2H, HPh), 7.49-
7.45 (m, 2H, HPh), 7.45-7.39 (m, 1H, HPh), 6.80 (s, 1H, H5), 6.46 (d, J = 4.0 Hz, 1H,
H1’), 5.67 (dd, J1 = 1.0 Hz, J2 = 4.0 Hz, 1H, H2’), 5.11 (d, J = 1.0 Hz, 1H, H3’), 4.54
(dd, J1 = 7.0 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.43 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a),
4.35-4.29 (m, 1H, H4’), 2.17 (s, 6H, 2 x MeAc), 1.92 (s, 3H, MeAc) 13C NMR (125
MHz, CDCl3): 171.9, 170.6, 169.6, 168.1, 156.1, 154.1, 136.5, 129.9, 128.9, 128.1,
124.9, 107.9, 97.5, 86.9, 81.6, 76.4, 73.9, 62.8, 20.8, 20.6, 20.4. IR (KBr cm-1): 3475,
3088, 3040, 2936, 1757, 1679, 1610, 1574, 1490, 1421, 1387, 1373, 1351, 1236, 1207,
1179, 1126, 1107, 1048, 1005; HRMS (ESI): m/z [M+H]+ calcd for C23H23N2O9:
471.13981, found: 471.13988.
3-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-6-(3-chlorophenyl)furo[2,3-
20
d]pyrimidin-2(3H)-one (9b). Yield: 70%, white crystals, mp.: 213.0-214.0 °C. [α]D
1
+81.3 (c 1.0, DMSO); H NMR (500 MHz, CDCl3): 8.34 (s, 1H, H4), 7.77-7.73 (m,
1H, HAr), 7.68-7.63 (m, 1H, HAr), 7.43-7.34 (m, 2H, HAr), 6.83 (s, 1H, H5), 6.45 (d, J
= 3.5 Hz, 1H, H1’), 5.67 (dd, J1 = 1.5 Hz, J2 = 3.5 Hz, 1H, H2’), 5.11 (d, J = 1.5 Hz, 1H,
H3’), 4.55 (dd, J1 = 7.0 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.43 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz,
10

ACCEPTED MANUSCRIPT
1H, H5’a), 4.34-4.29 (m, 1H, H4’), 2.170 (s, 3H, MeAc), 2.166 (s, 3H, MeAc), 1.93 (s,
3H, MeAc) 13C NMR (125 MHz, CDCl3): 171.8, 170.7, 169.6, 168.0, 154.5, 154.0,
137.2, 135.1, 130.3, 129.9, 129.8, 124.9, 123.0, 107.6, 98.8, 87.1, 81.7, 76.4, 73.9,
62.8, 20.8, 20.7, 20.4. HRMS (ESI): m/z [M+H]+ calcd for C23H2235ClN2O9:
505.10083, found: 505.10046.
3-(2,3,5-Tri-O-acetyl-β-
D
-arabinofuranosyl)-6-(4-chlorophenyl)furo[2,3-
20
d]pyrimidin-2(3H)-one (9c). Yield: 74%, colorless oil. [α]D +75.3 (c 1.0, DMSO);
1H NMR (500 MHz, CDCl3): 8.30 (s, 1H, H4), 7.71 (d, J = 8.5 Hz, 2H, HAr), 7.45 (d, J
= 8.5 Hz, 2H, HAr), 6.79 (s, 1H, H5), 6.45 (d, J = 3.5 Hz, 1H, H1’), 5.73-5.62 (m, 1H,
H2’), 5.15-5.05 (m, 1H, H3’), 4.55 (dd, J1 = 7.5 Hz, J2 = 11.5 Hz, 1H, H5’b), 4.42 (dd, J1
= 3.5 Hz, J2 = 11.5 Hz, 1H, H5’a), 4.36-4.26 (m, 1H, H4’), 2.170 (s, 3H, MeAc), 2.166
13
(s, 3H, MeAc), 1.92 (s, 3H, MeAc). C NMR (125 MHz, CDCl3): 171.9, 170.7, 169.7,
168.1, 155.0, 154.0, 136.8, 135.9, 129.3, 126.7, 126.2, 107.8, 98.0, 87.0, 81.7, 76.4,
73.9, 62.8, 20.9, 20.7, 20.5; HRMS (ESI): m/z [M+H]+ calcd for C23H2235ClN2O9:
505.10083, found: 505.10052.
3-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-6-(3,4-dichlorophenyl)furo[2,3-
d]pyrimidin-2(3H)-one (9d) . Yield: 72%, colorless oil. [α]D20 +71.7 (c 1.0, DMSO);
1H NMR (500 MHz, CDCl3): 8.35 (s, 1H, H4), 7.85 (d, J=2.0 Hz, 1H, HAr), 7.61 (dd,
J1 = 2.0 Hz, J2 = 8.5 Hz, 1H, HAr), 7.53 (d, J = 8.5 Hz, 1H, HAr), 6.83 (s, 1H, H5), 6.45
(d, J = 3.5 Hz, 1H, H1’), 5.66 (dd, J1 = 1.5 Hz, J2 = 3.5 Hz, 1H, H2’), 5.10 (d, J = 1.5
Hz, 1H, H3’), 4.56 (dd, J1 = 7.0 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.42 (dd, J1 = 4.0 Hz, J2 =
12.0 Hz, 1H, H5’a), 4.35-4.29 (m, 1H, H4’), 2.17 (s, 3H, MeAc), 2.16 (s, 3H, MeAc),
13
1.92 (s, 3H, MeAc). C NMR (125 MHz, CDCl3): 171.8, 170.7, 169.7, 168.0, 153.9,
153.5, 137.4, 133.9, 133.5, 131.1, 128.1, 126.7, 124.0, 107.4, 99.1, 87.1, 81.8, 76.4,
73.9, 62.8, 20.9, 20.7, 20.5. HRMS (ESI): m/z [M+H]+ calcd for C23H2035Cl2N2O9:
539.06186, found: 539.06132.
3-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-6-(2-pyridyl)furo[2,3-d]pyrimidin-
2(3H)-one (9e). Yield: 56%, white crystals, mp.: 175.7-176.7 °C. [α]D20 +86.3 (c 1.0,
1
DMSO); H NMR (500 MHz, CDCl3): 8.69-8.64 (m, 1H, HAr), 8.39 (s, 1H, H4), 7.87-
7.80 (m, 1H, HAr), 7.34-7.29 (m, 1H, HAr), 7.26 (s, 1H, H5), 6.47 (d, J = 4.0 Hz, 1H,
H1’), 5.67 (dd, J1 = 1.5 Hz, J2 = 4.0 Hz, 1H, H2’), 5.12 (d, J = 1.5 Hz, 1H, H3’), 4.53
11

ACCEPTED MANUSCRIPT
(dd, J1 = 6.5 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.45 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a),
4.36-4.30 (m, 1H, H4’), 2.17 (s, 3H, MeAc), 2.16 (s, 3H, MeAc), 1.92 (s, 3H, MeAc). 13C
NMR (125 MHz, CDCl3): 172.0, 170.5, 169.6, 168.1, 155.2, 154.0, 150.1, 147.1,
137.9, 137.1, 124.0, 120.2, 107.5 , 101.1, 86.9, 81.6, 76.3, 73.9, 62.7, 20.8, 20.7, 20.4;
HRMS (ESI): m/z [M+H]+ calcd for C22H22N3O9: 472.13506, found: 472.13462.
3-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-6-(3-hydroxyphenyl)furo[2,3-
d]pyrimidin-2(3H)-one (9f). Yield: 43%, yellow crystals, mp.: 210.0-211.0 °C.
1
[α]D20 +80.3 (c 1.0, DMSO); H NMR (500 MHz, CDCl3): 8.30 (s, 1H, H4), 7.75 (bs,
1H, OH), 7.59-7.55 (m, 1H, HAr), 7.31-7.22 (m, 2H, HAr), 6.97-6.92 (m, 1H, HAr),
6.77 (s, 1H, H5), 6.47 (d, J = 4.0 Hz, 1H, H1’), 5.69 (dd, J1 = 1.5 Hz, J2 = 4.0 Hz, 1H,
H2’), 5.11 (d, J = 1.5 Hz, 1H, H3’), 4.53 (dd, J1 = 6.5 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.43
(dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a), 4.35-4.30 (m, 1H, H4’), 2.17 (s, 3H, MeAc),
13
2.16 (s, 3H, MeAc), 1.90 (s, 3H, MeAc). C NMR (125 MHz, CDCl3): 172.0, 170.8,
169.8, 168.2, 157.3, 156.7, 154.8, 136.3, 130.1, 129.2, 117.6, 116.8, 112.3, 108.6,
97.7, 87.1, 81.7, 76.4, 74.0, 62.8, 20.9, 20.7, 20.4. HRMS (ESI): m/z [M+H]+ calcd
for C23H23N2O10: 487.13472, found: 487.13416.
3-(2,3,5-Tri-O-acetyl-β-
D
-arabinofuranosyl)-6-(4-n-pentylphenyl)furo[2,3-
20
d]pyrimidin-2(3H)-one (9g). Yield: 54%, colorless oil. [α]D +73.0 (c 1.0, DMSO);
1H NMR (500 MHz, CDCl3): 8.30 (s, 1H, H4), 7.69 (d, J = 8.0 Hz, 2H, CHAr), 7.26 (d,
J = 8.0 Hz, 2H, CHAr), 6.73 (s, 1H, H5), 6.46 (d, J = 3.5 Hz, 1H, H1’), 5.66 (dd, J1 =
1.5 Hz, J2=3.5 Hz, 1H, H2’), 5.11 (d, J = 1.5 Hz, 1H, H3’), 4.53 (dd, J1 = 6.5 Hz,
J2=12.0 Hz, 1H, H5’b), 4.44 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a), 4.36-4.30 (m, 1H,
H4’), 2.65 (t, J = 7.5 Hz, 2H, CH2chain), 2.16 (s, 6H, MeAc), 1.92 (s, 3H, MeAc), 1.64 (q,
J = 7.5 Hz, 2H, CH2chain), 1.39-1.29 (m, 4H, 2 x CH2chain), 0.90 (t, J = 7.0 Hz, 3H,
CH3chain). 13C NMR (125 MHz, CDCl3): 172.0, 170.6, 169.6, 168.0, 156.4, 154.1,
145.3, 136.0, 129.0, 125.6, 125.0, 108.1, 96.5, 86.9, 81.5, 76.4, 73.9, 62.8, 35.8, 31.4,
30.8, 22.4, 20.8, 20.6, 20.4, 13.9. HRMS (ESI): m/z [M+H]+ calcd for C28H32N2O9:
541.21806, found: 541.21734.
3-(2,3,5-Tri-O-acetyl-β-
D-arabinofuranosyl)-6-(3-fluorophenyl)furo[2,3-
20
d]pyrimidin-2(3H)-one (9h). Yield: 43%, white crystals, mp.: 226.4-227.0 °C. [α]D
+75.0 (c 1.0, DMSO); 1H NMR (500 MHz, CDCl3): 8.34 (s, 1H, H4), 7.57 (d, J = 7.5
12

ACCEPTED MANUSCRIPT
Hz, 1H, HAr), 7.49-7.38 (m, 2H, HAr), 7.11 (dt, J1 = 2.0 Hz, J2 = 8.5 Hz, 1H, HAr), 6.83
(s, 1H, H5), 6.46 (d, J = 3.5 Hz, 1H, H1’), 5.67 (dd, J1 = 1.5 Hz, J2 = 3.5 Hz, 1H, H2’),
5.11 (d, J = 1.5 Hz, 1H, H3’), 4.55 (dd, J1 = 7.0 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.43 (dd,
J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a), 4.35-4.30 (m, 1H, H4’), 2.170 (s, 3H, MeAc),
13
2.166 (s, 3H, MeAc), 1.93 (s, 3H, MeAc). C NMR (125 MHz, CDCl3): 171.8, 170.7,
169.6, 168.0, 163.0 (d, J=246.6 Hz), 154.7 (d, J=3.4 Hz), 154.0, 137.1, 130.7 (d,
J=8.3 Hz), 130.2 (d, J=8.8 Hz), 120.7 (d, J=2.9 Hz), 116.8 (d, J=21.5 Hz), 111.9 (d,
J=23.9 Hz), 107.6, 98.7, 87.0, 81.7, 76.4, 73.9, 62.8, 20.9, 20.7, 20.4.19F NMR (471
MHz, CDCl3): −111.98. HRMS (ESI): m/z [M+H]+ calcd for C23H22N2O9F:
489.13038, found: 489.13047.
3-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-6-(3,5-difluorophenyl)furo[2,3-
20
d]pyrimidin-2(3H)-one (9i).Yield: 66%, white crystals, mp.: 224.8-225.6 °C. [α]D
1
+82.2 (c 1.0, DMSO); H NMR (500 MHz, CDCl3): 8.39 (s, 1H, H4), 7.33-7.25 (m,
2H, HAr), 6.93-6.82 (m, 2H, H5+HAr), 6.45 (d, J = 3.5 Hz, 1H, H1’), 5.67 (dd, J1 = 1.5
Hz, J2 = 3.5 Hz, 1H, H2’), 5.10 (d, J = 1.5 Hz, 1H, H3’), 4.56 (dd, J1 = 7.0 Hz, J2 = 12.0
Hz, 1H, H5’b), 4.42 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a), 4.35-4.29 (m, 1H, H4’),
2.173 (s, 3H, MeAc), 2.165 (s, 3H, MeAc), 1.93 (s, 3H, MeAc).13C NMR (125 MHz,
CDCl3): 171.7, 170.7, 169.6, 168.0, 163.3 (dd, J1=12.7 Hz, J2=249.5 Hz), 153.9,
153.4 (t, J~3.6 Hz), 137.9, 131.1 (t, J~10.5 Hz), 108.0 (m), 107.3, 105.1 (d, J=25.4
Hz), 100.0, 87.1, 81.8, 76.4, 73.9, 62.8, 20.9, 20.7, 20.4; 19F NMR (471 MHz,
CDCl3): −108.28. HRMS (ESI): m/z [M+H]+ calcd for C23H21N2O9F2: 507.12096,
found: 507.12116.
3-(2,3,5-Tri-O-acetyl-β-
D
-arabinofuranosyl)-6-(4-trifluorometoxyphenyl)furo[2,3-
20
d]pyrimidin-2(3H)-one (9j). Yield: 43%, colorless oil. [α]D +56.6 (c 1.0, DMSO);
1H NMR (500 MHz, CDCl3): 8.34 (s, 1H, H4), 7.82-7.76 (m, 2H, HAr), 7.31 (d, J=8.5
Hz, 2H, HAr), 6.82 (s, 1H, H5), 6.46 (d, J=3.5 Hz, 1H, H1’), 5.67 (dd, J1=1.0 Hz,
J2=3.5 Hz, 1H, H2’), 5.11 (d, J=1.0 Hz, 1H, H3’), 4.55 (dd, J1=7.0 Hz, J2=11.5 Hz, 1H,
H5’a), 4.43 (dd, J1=4.0 Hz, J2=11.5 Hz, 1H, H5’b), 4.35-4.29 (m, 1H, H4’), 2.169 (s, 3H,
13
MeAc), 2.167 (s, 3H, MeAc), 1.93 (s, 3H, MeAc) C NMR (125 MHz, CDCl3): 171.9,
170.6, 169.6, 168.0, 154.6, 154.0, 150.0 (q, J = 1.9 Hz), 137.0, 126.8, 126.5, 121.3,
120.3 (q, J = 257.8 Hz), 107.6, 98.3, 87.0, 81.7, 76.4, 73.9, 62.8, 20.8, 20.6, 20.4; 19F
13

ACCEPTED MANUSCRIPT
NMR (471 MHz, CDCl3): −58.13. HRMS (ESI): m/z [M+H]+ calcd for
C24H21N2O10F3: 555.12211, found: 555.12207.
3-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-6-(methoxymethyl)furo[2,3-
d]pyrimidin-2(3H)-one (9k). Yield: 40%, yellow crystals, mp.: 108.5-110.1 °C.
[α]D20 +106.8 (c 1.0, DMSO); 1H NMR (500 MHz, CDCl3): 8.27 (s, 1H, H4), 6.50 (s,
1H, H5), 6.43 (d, J = 4.0 Hz, 1H, H1’), 5.65 (dd, J1 = 1.5 Hz, J2 = 4.0 Hz, 1H, H2’),
5.09 (d, J = 1.5 Hz, 1H, H3’), 4.51 (dd, J1 = 7.0 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.45 (s,
2H, CH2MOM), 4.42 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a), 4.33-4.27 (m, 1H, H4’),
3.48 (s, 3H, MeMOM), 2.16 (s, 3H, MeAc), 2.15 (s, 3H, MeAc), 1.91 (s, 3H, MeAc). 13C
NMR (125 MHz, CDCl3): 172.1, 170.6, 169.6, 168.0, 155.1, 154.0, 137.2, 106.6,
102.2, 86.9, 81.6, 76.4, 73.9, 66.5, 62.8, 58.9, 20.8, 20.7, 20.4; HRMS (ESI): m/z
[M+H]+ calcd for C20H24N2O9: 439.13472, found: 439.13485.
3-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-6-(3-cyanopropyl)furo[2,3-
d]pyrimidin-2(3H)-one (9l). Yield: 65%, yellow crystals, mp.: 107.8 °C
(decomposition). [α]D20 +105.6 (c 1.0, DMSO); 1H NMR (500 MHz, CDCl3): 8.21 (s,
1H, H4), 6.42 (d, J = 3.5 Hz, 1H, H1’), 6.32 (s, 1H, H5), 5.65 (dd, J1 = 1.5 Hz, J2 = 3.5
Hz, 1H, H2’), 5.09 (d, J = 1.5 Hz, 1H, H3’), 4.51 (dd, J1 = 7.0 Hz, J2 = 12.0 Hz, 1H,
H5’b), 4.41 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a), 4.33-4.27 (m, 1H, H4’), 2.88 (d, J
= 7.0 Hz, 2H, CH2Pr), 2.47 (d, J = 7.0 Hz, 2H, CH2Pr), 2.16 (s, 3H, MeAc), 2.15 (s, 3H,
MeAc), 2.12 (quintet, J = 7.0 Hz, 2H, CH2Pr), 1.92 (s, 3H, MeAc). 13C NMR (125 MHz,
CDCl3): 172.0, 170.6, 169.6, 168.0, 156.9, 154.0, 136.3, 118.6, 106.9, 100.6, 86.8,
81.5, 76.3, 73.8, 62.8, 27.0, 22.6, 20.8, 20.7, 20.4, 16.5. HRMS (ESI): m/z [M+H]+
calcd for C21H24N3O9: 462.15071, found: 462.15022.
3-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-6-(cyclopropyl)furo[2,3-d]pyrimidin-
2(3H)-one (9m). Yield: 31%, white crystals, mp.:161.8-163.1 °C. [α]D20 +102.1 (c 1.0,
DMSO); 1H NMR (500 MHz, CDCl3): 8.07 (s, 1H, H4), 6.43 (d, 1H, J = 3.5 Hz, H1’),
6.15 (s, 1H, H5), 5.63 (d, J = 3.5 Hz, 1H, H2’), 5.09 (s, J = 1.5 Hz, 1H, H3’), 4.49 (dd,
J1 = 7.0 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.42 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a),
4.32-4.23 (m, 1H, H4’), 2.16 (s, 3H, MeAc), 2.15 (s, 3H, MeAc), 1.86-1.97 (m, 4H,
13
MeAc + CHcPr), 1.08-0.97 (m, 4H, 2 x CH2cPr). C NMR (125 MHz, CDCl3): 171.7,
170.5, 169.6, 168.0, 161.0, 154.1, 134.4, 107.8, 97.0, 86.7, 81.3, 76.3, 73.8, 62.8, 20.8,
14

ACCEPTED MANUSCRIPT
20.6, 20.4, 9.3, 7.30, 7.27. HRMS (ESI): m/z [M+H]+ calcd for C21H24N3O9:
435.13967, found: 435.13981.
3-(2,3,5-Tri-O-acetyl-β-
D
-arabinofuranosyl)-6-benzylfuro[2,3-d]pyrimidin-2(3H)-
20
one (9n). Yield: 32%, yellow crystals, mp.: 87.6-90.7 °C. [α]D +85.4 (c 1.0,
DMSO); 1H NMR (500 MHz, CDCl3): 8.13 (s, 1H, H4), 7.33-7.26 (m, 2H, HAr), 7.40-
7.23 (m, 3H, HAr), 6.41 (d, J = 3.5 Hz, 1H, H1’), 6.09 (s, 1H, H5), 5.63 (dd, J1 = 1.0 Hz,
J2 = 3.5 Hz, 1H, H2’), 5.07 (d, J = 1.0 Hz, 1H, H3’), 4.46 (dd, J1 = 7.0 Hz, J2 = 12.0 Hz,
1H, H5’b), 4.39 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 1H, H5’a), 4.30-4.24 (m, 1H, H4’), 3.99
(s, 1H, CH2Bn), 2.15 (s, 3H, MeAc), 2.12 (s, 3H, MeAc), 1.90 (s, 3H, MeAc). 13C NMR
(125 MHz, CDCl3): 172.2, 170.6, 169.6, 168.1, 158.8, 154.1, 135.8, 135.0, 129.0,
128.8, 127.3, 107.3, 100.0, 86.7, 81.4, 76.3, 73.8, 62.7, 34.8, 20.8, 20.6, 20.4. HRMS
(ESI): m/z [M+H]+ calcd for C24H24N2O9: 485.15546, found: 485.15514.
3-(2,3,5-Tri-O-acetyl-β-
D
-arabinofuranosyl)-6-n-octylfuro[2,3-d]pyrimidin-2(3H)-
20
one (9o). Yield: 41%, white crystals, mp.: 118.5-119.5°C. [α]D +89.0 (c 1.0,
DMSO); 1H NMR (500 MHz, CDCl3): 8.12 (s, 1H, H4), 6.44 (d, J = 3.5 Hz, 1H, H1’),
6.17 (s, 1H, H5), 5.64 (dd, J1 = 1.5 Hz, J2 = 3.5 Hz, 1H, H2’), 5.09 (d, J = 1.5 Hz, 1H,
H3’), 4.49 (dd, J1 = 7.0 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.41 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz,
1H, H5’a), 4.32-4.25 (m, 1H, H4’), 2.66 (t, J = 7.5 Hz, 2H, CH2Oct), 2.16 (s, 3H, MeAc),
2.15 (s, 3H, MeAc), 1.91 (s, 3H, MeAc), 1.74-1.65 (m, 2H, CH2Oct), 1.43-1.22 (m, 10H,
5 x CH2Oct), 0.88 (t, J = 7.0 Hz, 3H, MeOct); 13C NMR (125 MHz, CDCl3): 171.0,
170.6, 169.6, 168.1, 160.3, 154.2, 135.3, 107.5, 98.8, 86.7, 81.4, 76.4, 73.9, 62.8, 31.8,
29.2, 29.1, 29.0, 28.3, 26.6, 22.6, 20.8, 20.7, 20.4, 14.0; HRMS (ESI): m/z [M+H]+
calcd for C25H35N2O9: 507.23371, found: 507.23298.
3-(2,3,5-Tri-O-acetyl-β-D-arabinofuranosyl)-6-(9-hydroksy-n-nonyl)octylfuro[2,3-
d]pyrimidin-2(3H)-one (9p). Yield: 56%, colorless oil. [α]D20 +83.8 (c 1.0, DMSO);
1H NMR (500 MHz, CDCl3): 8.12 (s, 1H, H4), 6.44 (d, J = 3.5 Hz, 1H, H1’), 6.17 (t, J1
= 1.0 Hz, 1H, H5), 5.64 (dd, J1 = 1.5 Hz, J2 = 3.5 Hz, 1H, H2’), 5.09 (t, J = 1.5 Hz, 1H,
H3’), 4.50 (dd, J1 = 7.0 Hz, J2 = 12.0 Hz, 1H, H5’b), 4.41 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz,
1H, H5’a), 4.32-4.25 (m, 1H, H4’), 3.64 (t, J = 6.5 Hz, 2H, CH2Non), 2.66 (t, J = 7.5 Hz,
2H, CH2Non), 2.16 (s, 3H, MeAc), 2.15 (s, 3H, MeAc), 1.91 (s, 3H, MeAc), 1.70 (quintet,
J = 7.5 Hz, 2H, CH2Non), 1.62-1.52 (m, 2H, CH2Non), 1.42-1.28 (m, 10H, 5 x CH2Non);
15

ACCEPTED MANUSCRIPT
13C NMR (125 MHz, CDCl3): 172.2, 170.6, 169.7, 168.1, 160.5, 154.2, 135.2, 107.5,
98.6, 86.8, 81.4, 76.4, 73.9, 62.8, 62.9, 32.7, 29.3, 29.1, 28.9, 28.3, 26.6, 25.7, 20.8,
20.7, 20.4; HRMS (ESI): m/z [M+H]+ calcd for C26H37N2O10: 537.24427, found:
537.24361.
General procedures for the synthesis of deprotected 6-aryl/6-heteroaryl 3H-
pyrrolo[2,3-d]pyrimidin-2(7H)-one arabinosides 5
Procedure A was applied for the synthesis of 5a-5j compounds, while procedure B
was applied for the synthesis of 5k-5p compounds.
A: Protected furo[2,3-d]pyrimidin-2(3H)-one nucleosides 9 were dissolved in the 7N
methanolic ammonia (10 mL by 1 mmol of nucleoside) in a microwave tube and the
reaction mixture was heated in 60-65 °C in the microwave synthesizer (80 Watt) for
24-48 h. Then the reaction mixture was moved to the round-bottom flask, silica gel
was added and solvent was evaporated under the reduced pressure. The final
compounds were purified by column chromatography on silica gel using 10%, then
20% methanol in chloroform as a mobile phase.
B: Protected furo[2,3-d]pyrimidin-2(3H)-one nucleosides 9 were dispersed in the 25%
aquaous ammonia (24 mL by 1 mmol of nucleoside) and stirred at room temperature
for 1 – 4 days. In case of the compound 5p, the reaction mixture was heated at 50-
60 °C and aditional portion of ammonia was added. After completion of the reaction,
volatiles were evaporated under the reduced pressure and final compounds were
purified by column chromatography on silica gel using 10%, then 20% methanol in
chloroform as a mobile phase.
3-(β-D-Arabinofuranosyl)-6-phenyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one (5a).
20
1
Yield: 90%, yellow crystals, m.p. 165.5- 167.0 °C. [α]D +77.8 (c 1.0, DMSO); H
NMR (500 MHz, CD3OD): 8.62 (s, 1H, H4), 7.72-7.68 (m, 2H, HAr), 7.42-7.38 (t,
J=7.3 Hz, 2H, HAr), 7.35-7.31 (m, 1H, HAr), 6.65 (s, 1H, H5), 6.39 (d, J=3.5 Hz, 1H,
H1’), 4.38 (dd, J1=2.0 Hz, J2=3.5 Hz, 1H, H2’), 4.16 (t, J=2.0 Hz, 1H, H3’), 4.10-4.06
(m, 1H, H4’), 3.90 (d, J=4.5 Hz, 2H, H5’). 13C NMR (125 MHz, CD3OD): 160.5, 156.9,
141.8, 139.6, 132.0, 130.0, 129.6, 126.3, 111.5, 98.2, 90.2, 87.4, 78.3, 76.5, 62.9. IR
(KBr cm-1): 3700-2800, 1658, 1618, 1586, 1571, 1554, 1492, 1448, 1412, 1384, 1348,
16

ACCEPTED MANUSCRIPT
1253, 1198, 1107, 1055; HRMS (ESI): m/z [M+H]+ calcd for C17H18N3O5: 344.12308,
found: 344.12350.
3-(β-D-Arabinofuranosyl)-6-(3-chlorophenyl)-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-
20
one (5b). Yield: 32%, yellow crystals, m.p. 212.0-213.3 °C. [α]D +70.2 (c 1.0,
1
DMSO); H NMR (500 MHz, DMSO-d6): 11.80 (s, 1H, NH), 8.46 (s, 1H, H4), 7.93-
7.91 (m, 1H, HAr), 7.77 (dq, J1=1.0 Hz, J2=8.0 Hz, 1H, HAr), 7.46 (t, J=8.0 Hz, 1H,
HAr), 7.38 (dq, J1=1.0 Hz, J2=8.0 Hz, 1H, HAr), 6.88 (s, 1H, H5), 6.23 (d, J=3.5 Hz,
1H, H1’), 5.51 (d, J=4.0 Hz, 1H, OH2’), 5.47 (d, J=5.0 Hz, 1H, OH3’), 5.15 (t, J=5.5
Hz, 1H, OH5’), 4.16-4.11 (m, 1H, H2’), 3.99-3.95 (m, 1H, H3’), 3.89 (dt, J1=2.5 Hz,
J2=6.5 Hz, 1H, H4’), 3.74-3.63 (m, 2H, H5’). 13C NMR (125 MHz, DMSO-d6): 159.7,
153.8, 139.0, 137.2, 133.8, 132.8, 130.7, 127.8, 124.5, 123.5, 108.1, 98.6, 87.9, 85.9,
76.4, 74.5, 61.2. HRMS (ESI): m/z [M+H]+ calcd for C17H17N3O5Cl: 378.08512,
found: 378.08490.
3-(β-D-Arabinofuranosyl)-6-(4-chlorophenyl)-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-
20
one (5c). Yield: 32%, yellow crystals, m.p. 197.1-198.3 °C. [α]D +67.4 (c 1.0,
1
DMSO); H NMR (500 MHz, DMSO-d6): 11.80 (s, 1H, NH), 8.44 (s, 1H, H4), 7.77-
7.80 (m, 2H, HAr), 7.53-7.47 (m, 2H, HAr), 6.80 (s, 1H, H5), 6.23 (d, J=3.5 Hz, 1H,
H1’), 5.52 (d, J=4.0 Hz, 1H, OH2’), 5.47 (d, J=5.5 Hz, 1H, OH3’), 5.16 (t, J=5.5 Hz,
1H, OH5’), 4.17-4.11 (m, 1H, H2’), 4.00-3.96 (m, 1H, H3’), 3.89 (dt, J1=2.5 Hz, J2=5.5
Hz, 1H, H4’), 7.72-3.64 (bt, 2H, H5’). 13C NMR (125 MHz, DMSO-d6): 159.8, 153.8,
138.8, 137.6, 132.6, 129.6, 128.9, 126.6, 108.3, 97.9, 87.9, 85.9, 76.5, 74.5, 61.2.
HRMS (ESI): m/z [M+H]+ calcd for C17H17N3O5Cl: 378.08512, found: 378.08462.
3-(β-D-Arabinofuranosyl)-6-(3,4-dichlorophenyl)-3H-pyrrolo[2,3-d]pyrimidin-
2(7H)-one (5d). Yield: 41%, yellow crystals, m.p. 226.5-227.4 °C. [α]D20 +84.0 (c 1.0,
1
DMSO); H NMR (500 MHz, DMSO-d6): 11.75 (wave, 1H, NH), 8.47 (s, 1H, H4),
8.09 (d, J=2.0 Hz, 1H, HAr), 7.80 (d, J1=2.0 Hz, J2=7.5 Hz, 1H, HAr), 7.67 (d, J=7.5
Hz, 1H, HAr), 6.92 (s, 1H, H5), 6.23 (d, 1H, J=3.5 Hz, 1H, H1’), 5.52 (wave, 2H,
OH2’+OH3’), 5.16 (wave, 1H, OH5’), 4.18-4.11 (m, 1H, H2’), 4.01-3.94 (m, 1H, H3’),
3.89 (dt, J1=2.5 Hz, J2=5.5 Hz, 1H, H4’), 3.68 (d, J=5.5 Hz, 2H, H5’). 13C NMR (125
MHz, DMSO-d6): 159.7, 153.7, 139.2, 137.4, 131.8, 131.4, 131.0, 130.2, 126.4, 125.0,
17

ACCEPTED MANUSCRIPT
108.2, 99.2, 87.9, 86.0, 76.4, 74.5, 61.2; HRMS (ESI): m/z [M+H]+ calcd for
C17H17N3O5Cl2: 412.04615, found: 412.04592.
3-(β-D-Arabinofuranosyl)-6-(2-pirydyl)-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one
(5e). Yield: 46%, yellow solid, m.p. 188.5-189.1 °C. [α]D20 +103.7 (c 1.0, DMSO); 1H
NMR (500 MHz, DMSO-d6): 11.75 (s, 1H, NH), 8.64-8.59 (m, 1H, HAr), 8.51 (s, 1H,
H4), 7.92 (dt, J1=1.0 Hz, J2=8.0 Hz, 1H, HAr), 7.89-7.83 (m, 1H, HAr), 7.32 (ddd,
J1=1.0 Hz, J2=4.5 Hz, J3=8.0 Hz, 1H, HAr), 7.00 (s, 1H, H5), 6.24 (d, J=3.5 Hz, 1H,
H1’), 5.51 (d, J=4.0 Hz, 1H, OH2’), 5.47 (d, J=5.5 Hz, 1H, OH3’), 5.13 (t, J=5.5 Hz,
1H, OH5’), 4.17-4.11 (m, 1H, H2’), 4.01-3.95 (m, 1H, H3’), 3.89 (dt, J1=2.5 Hz, J2=5.5
Hz, 1H, H4’), 3.74-3.64 (m, 2H, H5’). 13C NMR (125 MHz, DMSO-d6): 159.4, 153.8,
149.5, 148.0, 139.7, 138.7, 137.1, 122.8, 119.8, 108.2, 99.9, 87.9, 86.0, 76.5, 74.5,
61.3; HRMS (ESI): m/z [M+H]+ calcd for C16H16N4O5: 345.11935, found: 345.11775.
3-(β-D-Arabinofuranosyl)-6-(3-hydroksyphenyl)-3H-pyrrolo[2,3-d]pyrimidin-
20
2(7H)-one (5f). Yield: 74%, yellow solid, m.p. 231.5-232.4 °C. [α]D +84.0 (c 1.0,
1
DMSO); H NMR (500 MHz, DMSO-d6): 11.67 (s, 1H, NH), 9.60 (s, 1H, OH), 8.39
(s, 1H, C4), 7.35-7.14 (m, 3H, HAr), 6.82-6.73 (m, 1H, HAr), 6.65 (s, 1H, H5), 6.23 (d,
J=3.5 Hz, 1H, H1’), 5.50 (d, J=4.0 Hz, 1H, OH2’), 5.43 (d, J=5.5 Hz, 1H, OH3’), 5.13
(t, J=5.0 Hz, 1H, OH5’), 4.17-4.06 (m, 1H, H2’), 4.02-3.95 (m, 1H, H3’), 3.92-3.85 (m,
1H, H4’), 3.75-3.63 (m, 2H, H5’). 13C NMR (125 MHz, DMSO-d6): 159.8, 157.7,
153.9, 139.1, 138.3, 129.9, 116.0, 115.4, 111.9, 108.3, 96.9, 87.8, 85.9, 76.5, 74.5,
61.3; HRMS (ESI): m/z [M+H]+ calcd for C16H16N4O5: 360.11901, found: 360.11752.
3-(β-D-Arabinofuranosyl)-6-(4-n-pentylphenyl)-3H-pyrrolo[2,3-d]pyrimidin-
20
2(7H)-one (5g). Yield: 55%, yellow solid, m.p. 124.3-125.9 °C. [α]D +61.3 (c 1.0,
DMSO); 1H NMR (500 MHz, CD3OD): 8.58 (s, 1H, H4), 7.58 (d, J=8.0 Hz, 2H, HAr),
7.20 (d, J=8.0 Hz, 2H, HAr), 6.58 (s, 1H, H5), 6.39 (d, J=3.5 Hz, 1H, H1’), 4.38 (dd,
J1=3.5 Hz, J2=2.0 Hz, 1H, H2’), 4.16 (t, J=2.0 Hz, 1H, H3’), 4.11-4.05 (m, 1H, H4’),
3.90 (d, J=4.5 Hz, 2H, H5’), 2.61 (t, J=7.5 Hz, 2H, CH2), 1.62 (quintet, J=7.5 Hz, 2H,
13
CH2), 1.37-1.31 (m, 4H, 2 x CH2), 0.90 (t, J=7.0 Hz, 3H, Me). C NMR (125 MHz,
CD3OD): 160.5, 156.9, 144.9, 139.2, 130.0, 129.4, 126.2, 111.6, 97.4, 90.2, 87.3, 78.3,
76.5, 62.9, 36.6, 32.6, 32.2, 23.6, 14.4; HRMS (ESI): m/z [M+H]+ calcd for
C22H27N3O5: 414.20235, found: 414.20177.
18

ACCEPTED MANUSCRIPT
3-(β-D-Arabinofuranosyl)-6-(3-fluorophenyl)-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-
one (5h). Yield: 94%, yellow glass. [α]D20 +84.3 (c 1.0, DMSO); 1H NMR (500 MHz,
DMSO-d6): 11.81 (s, 1H, NH), 8.48 (s, 1H, C4), 7.74-7.63 (m, 2H, HAr), 7.52-7.42 (m,
1H, HAr), 7.19-7.11 (m, 1H, HAr), 6.86 (s, 1H, H5), 6.25 (d, J=3.5 Hz, 1H, H1’), 5.53
(d, J=4.0 Hz, 1H, OH2’), 5.50 (d, J=5.0 Hz, 1H, OH3’), 5.16 (t, J=5.5 Hz, 1H, OH5’),
4.20-4.12 (m, 1H, H2’), 4.05-3.96 (m, 1H, H3’), 3.94-3.86 (m, 1H, H4’), 3.81-3.75 (m,
2H, H5’). 13C NMR (125 MHz, DMSO-d6): 162.6 (d, J=243.0 Hz), 159.7, 153.9, 139.1,
137.6, 133.1 (d, J=8.4 Hz), 130.9 (d, J=8.6 Hz), 121.1 (d, J=2.3 Hz), 114.8 (d, J=21.3
Hz), 111.7 (d, J=23.1 Hz), 108.2, 98.6, 88.0, 86.0, 76.5, 74.6, 61.3; 19F NMR (471
MHz, DMSO-d6): −112.52. HRMS (ESI): m/z [M+H]+ calcd for
C17H17FN3O5: 326.11468, found: 326.11510.
3-(β-D-Arabinofuranosyl)-6-(3,5-difluorophenyl)-3H-pyrrolo[2,3-d]pyrimidin-
2(7H)-one (5i). Yield: 60%, yellow crystals, decomposition at ~159.7 °C. [α]D20 +77.7
(c 1.0, DMSO); 1H NMR (500 MHz, DMSO-d6): 11.81 (s, 1H, NH), 8.50 (s, 1H, C4),
7.58 (d, J=7.0 Hz, 2H, HAr), 7.21-7.15 (m, 1H, HAr), 6.98 (s, 1H, H5), 6.24 (d, J=3.5
Hz, 1H, H1’), 5.56-5.48 (m, 2H, OH2’+OH3’), 5.15 (t, J=5.5 Hz, 1H, OH5’), 4.21-4.13
(m, 1H, H2’), 4.02-3.96 (m, 1H, H3’), 3.94-3.87 (m, 1H, H4’), 3.75-3.64 (m, 2H, H5’).
13C NMR (125 MHz, DMSO-d6): 162.8 (dd, J1~13.9 Hz, J2~245.2 Hz), 159.6, 153.8,
139.6, 136.5, 134.1 (t, J~10.8 Hz), 108.0 (m), 103.2 (t, J~26.2 Hz), 99.9, 88.1, 86.0,
76.5, 74.5, 61.2’ 19F NMR (471 MHz, DMSO-d6): −109.61. HRMS (ESI): m/z
[M+H]+ calcd for C17H15F2N3O5: 380.10525, found: 380.10551.
3-(β-D-Arabinofuranosyl)-6-(4-trifluorometoxyphenyl)-3H-pyrrolo[2,3-
20
d]pyrimidin-2(7H)-one (5j). Yield: 41%, yellow solid, m.p. 170.8-173.1 °C. [α]D
1
+72.0 (c 1.0, DMSO); H NMR (500 MHz, CD3OD): 8.67 (s, 1H, H4), 7.86-7.80 (m,
2H, HAr), 7.35 (d, J=8.0 Hz, 2H, HAr), 6.74 (s, 1H, H5), 6.39 (d, J=3.5 Hz, 1H, H1’),
4.36 (dd, J1=2.5 Hz, J2=3.5 Hz, 1H, H2’), 4.14 (t, J=2.5 Hz, 1H, H3’), 4.08 (dd, J1=2.5
Hz, J2=4.5 Hz, 1H, H4’), 3.90 (d, J=4.5 Hz, 2H, H5’). 13C NMR (125 MHz, CD3OD):
160.7, 157.0, 150.4, 140.3, 140.2, 131.3, 128.0, 122.6, 111.3, 99.3, 90.3, 87.5, 78.3,
76.5, 62.9, quarternary carbon in CF3O group was not observed in carbon spectrum.
19F NMR (471 MHz, CD3OD): −59.45. HRMS (ESI): m/z [M+H]+ calcd for
C18H17F3N3O6: 428.10640, found: 428.10636.
19

ACCEPTED MANUSCRIPT
3-(β-D-Arabinofuranosyl)-6-methoxymethyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-
one (5k). Yield: 31%, white solid, m.p. 147.2 °C (decomposition). [α]D20 +99.9 (c 1.0,
DMSO); 1H NMR (500 MHz, CD3OD): 8.60 (s, 1H, H4), 6.37 (d, J=3.5 Hz, 1H, H1’),
6.29 (s, 1H, H5), 4.43 (s, 2H, CH2MOM), 4.33 (dd, J1=2.5 Hz, J2=3.5 Hz, 1H, H2’), 4.14
(t, J=2.5 Hz, 1H, H3’), 4.08 (dd, J1=2.5 Hz, J2=4.5 Hz, 1H, H4’), 3.87 (d, J=4.5 Hz, 2H,
H5’), 3.37 (s, 3H, MeMOM); 13C NMR (125 MHz, CD3OD): 160.0, 156.9, 140.1, 139.7,
110.4, 101.3, 90.1, 87.4, 78.3, 76.4, 67.9, 62.9, 58.3; HRMS (ESI): m/z [M+H]+ calcd
for C13H18N3O6: 312.11901, found: 312.11905.
3-(β-D-Arabinofuranosyl)-6-(3-cyanopropyl)-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-
20
one (5l). Yield: 51%, yellow crystals, m.p. 124.8-126.9 °C. [α]D +100.9 (c 1.0,
DMSO); 1H NMR (500 MHz, CD3OD): 8.51 (s, 1H, H4), 6.36 (d, J=4.0 Hz, 1H, H1’),
6.11 (s, 1H, H5), 4.35-4.31 (m, 1H, H2’), 4.15-4.11 (m, 1H, H3’), 4.08-4.03 (m, 1H,
H4’), 3.89-3.85 (m, 2H, H5’), 2.78 (t, J=7.5 Hz, 2H, CH2Pr); 2.50 (t, J=7.0 Hz, 2H,
CH2Pr); 2.06-1.97 (m, 2H, CH2Pr); 13C NMR (125 MHz, CD3OD): 160.0, 156.9,
138.7, 120.7, 110.9, 99.3, 90.0, 87.3, 78.2, 76.5, 62.8, 25.1, 27.8, 16.8; HRMS (ESI):
m/z [M+H]+ calcd for C15H19N4O5: 312.11901, found: 312.11905.
3-(β-D-Arabinofuranosyl)-6-cyclopropyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one
(5m). Yield: 46%, beige crystals, m.p. 148.8 °C (decomposition). [α]D20 +100.5 (c 1.0,
DMSO); 1H NMR (500 MHz, DMSO-d6): 11.06 (s, 1H, NH), 8.17 (s, 1H, C4), 6.19 (d,
J=3.5 Hz, 1H, H1’), 5.89 (s, 1H, H5), 5.47 (d, J=3.5 Hz, 1H, OH2’), 5.37 (d, J=5.0 Hz,
1H, OH3’), 5.11 (bs, 1H, OH5’), 4.11-4.04 (m, 1H, H2’), 3.96-3.91 (m, 1H, H3’), 3.87-
3.82 (m, 1H, H4’), 3.68-3.61 (m, 2H, H5’), 1.92-1.82 (m, 1H, CHcPr), 0.95-0.87 (m, 2H,
13
CH2cPr), 0.81-0.70 (m, 2H, CH2cPr). C NMR (125 MHz, DMSO-d6): 159.1, 153.9,
143.9, 136.0, 108.0, 94.4, 87.6, 85.7, 76.5, 74.5, 61.2, 9.1, 7.6, 7.4; HRMS (ESI): m/z
[M+H]+ calcd for C14H18N3O5: 308.12410, found: 308.12367.
3-(β-D-Arabinofuranosyl)-6-benzyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one (5n).
20
Yield: 24%, yellow crystals, m.p. 180.8 °C (decomposition). [α]D +78.9 (c 1.0,
1
DMSO); H NMR (500 MHz, DMSO-d6): 11.07 (s, 1H, NH), 8.26 (s, 1H, C4), 7.36-
7.26 (m, 4H, HPh), 7.26-7.18 (m, 5H, HPh), 6.20 (d, J=3.5 Hz, 1H, H1’), 5.91 (s, 1H,
H5), 5.46 (d, J=4.0 Hz, 1H, OH2’), 5.36 (d, J=5.0 Hz, 1H, OH3’), 5.12-5.03 (m, 1H,
20

ACCEPTED MANUSCRIPT
OH5’), 4.12-4.05 (m, 1H, H2’), 3.97-3.92 (m, 1H, H3’), 3.91-82 (m, 3H, H4’ + CH2Bn),
3.65 (m, 2H, H5’); 13C NMR (125 MHz, DMSO-d6): 159.3, 153.9, 140.6, 138.3, 137.1,
128.7, 128.4, 126.5, 107.8, 97.7, 87.7, 85.8, 76.5, 74.5, 61.2, 33.7; HRMS (ESI): m/z
[M+H]+ calcd for C14H18N3O5: 358.13975, found: 358.13915.
3-(β-D-Arabinofuranosyl)-6-n-octyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one (5o).
20
1
Yield: 63%, white crystals, m.p. 110.5-112.7 °C. [α]D +72.2 (c 1.0, DMSO); H
NMR (500 MHz, DMSO-d6): 11.06 (s, 1H, NH), 8.22 (s, 1H, C4), 6.20 (d, J=3.5 Hz,
1H, H1’), 5.92 (s, 1H, H5), 5.46 (d, J=4.0 Hz, 1H, OH2’), 5.36 (d, J=5.5 Hz, 1H, OH3’),
5.09 (t, J=5.0 Hz, 1H, OH5’), 4.11-4.05 (m, 1H, H2’), 3.98-3.91 (m, 1H, H3’), 3.88-
3.82 (m, 3H, H4’), 3.65 (t, J=5.0 Hz, 2H, H5’); 2.51 (t, J=7.5 Hz, 2H, CH2Oct), 1.65-
1.55 (m, 2H, CH2Oct), 1.34-1.18 (m, 10H, 5 x CH2Oct), 0.86 (t, J=7.0 Hz, 3H, MeOct);
13C NMR (125 MHz, DMSO-d6): 159.1, 153.9, 141.8, 136.5, 107.9, 96.3, 87.6, 85.7,
76.5, 74.5, 61.2, 31.3, 28.7, 28.6, 28.5, 27.6, 27.5, 22.1, 13.9; HRMS (ESI): m/z
[M+H]+ calcd for C19H29N3O5: 380.21800, found: 380.21783.
3-(β-D-Arabinofuranosyl)-6-(9-hydroksy-n-nonyl)-3H-pyrrolo[2,3-d]pyrimidin-
2(7H)-one (5p).
20
Yield: 32%, white crystals, m.p. 196.3 °C (decomposition). [α]D +88.6 (c 1.0,
DMSO); 1H NMR (500 MHz, DMSO-d6): 11.07 (s, 1H, NH), 8.22 (s, 1H, C4), 6.20 (d,
J=4.0 Hz, 1H, H1’), 5.92 (s, 1H, H5), 5.47 (d, J=4.0 Hz, 1H, OH2’), 5.37 (d, J=5.5 Hz,
1H, OH3’), 5.10 (t, J=5.5 Hz, 1H, OH5’), 4.06-4.12 (m, 1H, H2’), 3.98-3.92 (m, 1H,
H3’), 3.88-3.82 (m, 3H, H4’), 3.66 (t, J=5.5 Hz, 2H, H5’); 3.41-3.32 (m, 2H, CH2Non),
2.56-2.47 (m, 2H, CH2Non), 1.66-1.55 (m, 2H, CH2Non), 1.44-1.34 (m, 2H, CH2Non),
1.33-1.18 (m, 10H, 5 x CH2Non); 13C NMR (125 MHz, DMSO-d6): 159.1, 153.9, 141.9,
136.5, 108.0, 96.3, 87.6, 85.7, 76.5, 74.5, 61.2, 60.7, 32.5, 29.0, 28.7, 28.6, 27.6, 27.5,
25.5; HRMS (ESI): m/z [M+H]+ calcd for C20H31N3O6: 410.22856, found: 410.22831.
4.2 Biology - Cytotoxic Assays
Cell culture: HL-60 human promyelocytic leukemia cells and Jurkat E6.1 human
acute T cell leukemia cells were obtained from the American Type Culture Collection
(Rockville, Maryland, U.S.A.) and maintained in the Cell Culture Collection of the
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences
(IIET, PAS), Wroclaw, Poland. Cells were maintained in RPMI-1640 GLUTAMAX
21

ACCEPTED MANUSCRIPT
(Gibco, Scotland, UK) medium containing 100 U/mL penicillin, 100 ꢀg/mL
streptomycin (both from Polfa Tarchomin S.A., Warsaw, Poland) and supplemented
with 10% fetal bovine serum (Sigma-Aldrich, Germany). Medium for HL-60 cells
was additionally supplemented with 1 mM sodium pyruvate and 4.5 g/L glucose (both
from Sigma-Aldrich, Germany).
Antiproliferative assays: Cells were plated on 96-well plates (Corning B.V. New
York, USA) at a density of 1 × 104 cells per well in 100 ꢀL of culture medium
without FBS and antibiotics. After 24 h of incubation under standard conditions (37
°C in humid atmosphere with 5 % CO ), cells were treated with cytarabine analogues
2
suspended in 100 µL of culture medium at final concentrations: 100 – 10 – 1 – 0.1
ꢀg/mL. After additional 72 h an MTT assay was applied as described earlier.45 The
optical densities of the samples were measured on a Synergy H4 Hybrid Reader
(BioTek Instruments, USA). Two reference compounds were applied: cisplatin and
cytarabine.
5. Acknowledgements
This work was supported by the Polish Ministry of Science and Higher Education
(“Iuventus” grant No. IP2011 035571). The equipment used was sponsored in part by
the Centre for Preclinical Research and Technology (CePT), a project co-sponsored
by European Regional Development Fund and Innovative Economy, The National
Cohesion Strategy of Poland. We thank Jacek Olędzki for recording ES-MS spectra.
6. References
Revised order/numbering of references
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4
A. Hoshi, F. Kanzawa, K. Kuretani, T. Kanai, K. Kikugawa, M. Ichino Gann 1973,
64, 519-522.
5 A. Hoshi, F. Kanzawa, M. Iigo, K. Kuretani Gann 1975, 66, 539-546.
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6 J. H. Burchenal, T. O’Toole, K. Kalaher, J. Chrisholm Cancer Res. 1977, 37, 4098-
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H. Hirayama, K. Sugihara, K. Wakigawa, M. Iwamura, J. Hikita, H. Ohkuma,
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H. Hirayama, T. Sugihara, K. Sugihara, K. Wakigawa, M. Iwamura, H. Ohkuma,
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K. Sugihara, T. Sugihara, J. Hikita, H. Ohkuma, H. Hirayama Pharmacometrics
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H. Hirayama, T. Sugihara, K. Sugihara, K. Wakigawa, J. Hikita, H. Ohkuma
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11 J. Stentoft, Drug Saf. 1990, 5, 7-27
12 W. J. Baker, G. L. Royer Jr., R. B. Weiss J. Clin. Oncol. 1991, 9, 679-693.
13 M. Aoshima, S. Tsukagoshi, Y. Sakurai Cancer Res. 1977, 37, 2481-2486.
14 T. Ueda, T. Nakamura, S. Ando Cancer Res. 1983, 43, 3412-3416.
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A. Hoshi, M. Yoshida, F. Kanzawa, K. Kuretani, T. Kanai, M. Ichino. Chem.
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16 M. C. Wang, R. Sharma, A. Bloch Cancer Res. 1973, 33, 1265-1271.
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2009, 28, 695-712.
18 J. Woo, R. B. Meyer Jr., H. B. Gamper Nucleic Acids Res. 1996, 24, 2470-2475.
19 A. S. Wahba, M. J. Damha, R. H. E. Hudson Nucleic Acids Symp. Series 2008, 52,
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