Synthesis and antiproliferative activity of quinolone nucleosides against the human myelogenous leukemia K-562 cell line

Article abstract of DOI:10.1002/ardp.201300232

A set of 6-substituted quinolone nucleosides linked to aniline or phenol via N or O heteroatom-bridges presenting new compounds were synthesized by palladium-catalyzed Buchwald-Hartwig cross-coupling reactions. 6-Bromoquinolone nucleoside precursors, bein

Full text of DOI:10.1002/ardp.201300232

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
1
Full Paper
Synthesis and Antiproliferative Activity of Quinolone
Nucleosides Against the Human Myelogenous Leukemia K-562
Cell Line
Lena Wicke1, Joachim W. Engels1, Roberto Gambari2, and Antoine M. Saab3
1
Johann Wolfgang Goethe-Universität, Institute of Organic Chemistry and Chemical Biology, Frankfurt am Main,
Germany
2
Department of Life Science and Biotechnology, Section of Biochemistry and Molecular Biology, University of
Ferrara, Ferrara, Italy
3
Faculty of Sciences II, Chemistry Department, Lebanese University, Fanar, Beirut, Lebanon
A set of 6-substituted quinolone nucleosides linked to aniline or phenol via N or O heteroatom-bridges
presenting new compounds were synthesized by palladium-catalyzed Buchwald–Hartwig cross-
coupling reactions. 6-Bromoquinolone nucleoside precursors, being protected by either benzoyl or
TBDMS protecting groups on the ribose moiety, were subjected to different Buchwald–Hartwig
conditions as the key step. Defined deprotection steps led, in good yields, to the final target compounds
that carry, in position 3, either ester, acid, or amide functions. Thus, a series of novel quinolone
nucleoside derivatives was obtained via a convergent synthesis route. Biological tests in human chronic
myelogenous leukemia K562 cells exerted an efficient antiproliferative activity for two of them without
induction of differentiation. These novel nucleosides deserve further experiments to determine their
antiproliferative effects on other CML cell lines.
Keywords: Antiproliferative activity / Buchwald–Hartwig coupling / K562 cells / Microwave-assisted synthesis /
6-Substituted quinolone nucleosides
Received: June 19, 2013; Revised: June 19, 2013; Accepted: August 7, 2013
DOI 10.1002/ardp.201300232
Introduction
anti-CML molecules affecting differentiated functions [3, 7–9].
Differentiation of K-562 cells is associated with an increase of
expression of embryo–fetal globin genes, such as the z-, e-, and
g-globin genes [10]. Using this system, several anti-cancer
agents have also been characterized with respect to activation
of erythroid functions, such as mithramycin [11], cisplatin
[12], and tallimustine [13].
Nowadays, a variety of synthetic nucleosides have been
shown to exhibit antibacterial, antiviral, and anti-cancer
activity associated with interference of biological activity of
different molecular targets; several nucleoside analogs have
been demonstrated to control infections and cancer cell
growth in vitro and in vivo. Most synthetic efforts for nucleoside
analogs were directed at modifications on the 20-carbon. For
instance, among this class of molecules, the pyrimidine
nucleoside analog arabinosyl cytosine (ara-C) had activity in
acute myelogenous leukemia [14]. In addition, it should be
mentioned that most nucleoside-derived drugs used in anti-
cancer therapy [15, 16] are fluorinated pyrimidine nucleo-
sides. MCF7 cells, a suitable model of breast carcinoma, are
Chronic myelogenous leukemia (CML) is characterized by
increased growth of myeloid lines of cells in blood and bone
marrow. Genetically, CML is characterized by the presence of
a Philadelphia (Ph1) chromosome (22q-), which results from a
reciprocal translocation between chromosomes 9 and 22 [1, 2].
Anti-CML agents have been widely described and character-
ized, inducing antiproliferative effects on CML cell lines, in
some cases associated with induction of differentiation
properties, such as expression of embryo–fetal globin genes
and accumulation of embryionic and fetal hemoglobins [3–6].
Among the in vitro cellular systems employed for drug
screening of anti-CML molecules, K-562 cells have been object
of several investigations, allowing identification of several
Correspondence: Joachim W. Engels, Johann Wolfgang Goethe-
Universität, Institute of Organic Chemistry and Chemical Biology, Max-
von-Laue-Str. 7, 60438 Frankfurt am Main, Germany.
E-mail: joachim.engels@chemie.uni-frankfurt.de
Fax: þ49 7982 9148
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
L. Wicke et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
very sensitive to the treatment with 50-deoxy-5-fluorouridine
(50-DFUR) [17]. Furthermore, the development of gemcitabine,
a carbohydrate-modified analog of deoxycytidine [18], was
found to be promising, considering its effects against a variety
of solid tumors; this class of anti-cancer molecules may also
have therapeutic value in hematological malignancies [19]. In
parallel with the demonstration of the anti-cancer potential of
gemcitabine, investigations were concluded on a family of
purine nucleoside analogs, fludarabine, cladribine, and
pentostatin, which were found to retain major activity in
indolent B-cell malignancies. The mechanisms of action and
the clinical activities of all these agents have recently been
reviewed in depth [20, 21].
Quinolones are well known as antibacterial antibiotics [22]
like nalidixic acid [23], norfloxacine [24], ciprofloxacine [25],
and amifloxacine [26]. In addition, fluoroquinolones have
been used to treat bacterial infection including respiratory
and urinary infection [27]. Considering the fact that several
quinolones show anti-cancer activities [28], the first objective
of this work was to carry out synthesis of four 6-substituted
quinolone nucleoside compounds. The second objective of
this work was to study the antiproliferative activity of these 6-
substituted quinolone nucleosides on human CML cells [12]. A
third objective of our study was to verify the ability of the four
6-substituted quinolones to induce terminal erythroid
differentiation using the K562 human CML cell line as model
system [12, 13, 29].
Herein, we describe the synthesis and antiproliferative
activity of quinolone ribonucleosides on K562 leukemia cells.
This class of compounds combines the pharmacological
patterns of quinolones known as antibacterial and antiviral
substances as well as of natural nucleosides or nucleoside
analogs commonly used in chemotherapy [30–35]. The
natural base moiety is substituted for the quinolone
heterocycle aiming at additional activities via associated
effects of both structures. In fact, few studies about
synthesis [30, 36] and biological evaluation [31, 35] (e.g.,
against HIV reverse transcriptase [37] and integrase [38] or
HSV [39]) of quinolone nucleoside analogs have been reported
so far.
lone nucleoside (3) or (4). While the motif of 4-quinolone-3-
carboxylic acid was found to be pharmacologically interest-
ing [38, 45] and all reported compounds bear the free acid, we
prepared the related acid derivative but also intended to
modify this particular group for reasons of comparing.
Different standard deprotection steps resulted in free ribose
for all compounds, and either the free acid (11), the amide (10
and 12), or the ester (9) in the quinolone position 3.
As shown in Scheme 1, starting from 6-bromo quinolone
(quinolone synthesis reported for several times, e.g., originally
from Gould and Jacobs [46], prepared by us [40] via convenient
microwave-assisted synthesis modified according to Kidwai et
al. [47] and Koga et al. [24]) the first step consisted of
Vorbrüggen-glycosylation [48, 49]. These steps were carried
out on a large scale and led to protected 6-bromoquinolone
nucleoside (2) or (3), giving rise to a convergent synthesis
strategy as this is the precursor for all desired target
compounds. Vorbrüggen-glycosylation could be performed
either by conventional heating (3 h) or under microwave
irradiation (1 h) [40, 41], resulting in both cases in particular
nucleosides in very good yield. Buchwald–Hartwig coupling –
which has not been described for these nucleoside systems yet
– had to be established following different procedures in
dependence on the selected coupling partner (aniline or
phenol). In both cases, nucleoside (2) with acetyl-protected
sugar moiety turned out to be unsuitable because of poor
stability during Buchwald–Hartwig coupling conditions. In
contrary to aniline coupling, which was possible on benzoyl-
protected nucleoside (3) yielding the final product (10) in only
few steps, varied conditions required for phenol coupling
were found to be incompatible with benzoyl-protection. Thus,
use of base-stable silyl-protected nucleoside (4) was necessary.
Even if this afforded three more steps in the synthesis route,
standard deprotection and protection steps as well as smooth
Buchwald–Hartwig coupling with derivative (4) resulted in an
overall convenient synthesis strategy in good yields and high
quality for the final phenol-coupled products (11) and (12).
Additionally, this route also allowed the preparation of
aniline compound (9) still bearing the ester function but free
hydroxyl groups of ribose. This feature could not be obtained
via benzoyl-strategy used for (10) because the deprotection
step simultaneously cleaved the benzoyl groups and the ester
function in position 3 of quinolone.
Results and discussion
Chemistry: Synthesis of 6-substituted quinolone
ribonucleosides
Biological tests
Based on the microwave-assisted synthesis of 6-fluoroquino-
lone nucleoside investigated in our group [40, 41], we decided
to expand the modification pattern and to build up 6-
substituted quinolone nucleosides linked to aniline or phenol
via N- or O-heteroatom-bridge. This can be generally achieved
by palladium-catalyzed Buchwald–Hartwig cross-cou-
pling [42–44] starting from the fluoro-analog 6-bromoquino-
In order to evaluate the biological activity of the four 6-
substituted quinolone nucleoside compounds, chronic leuke-
mia K562 cells were seeded at initial cell concentration of
30,000 cells/mL and then cultured for 5 days in presence or
absence of compounds, employed at concentrations ranging
from 0.1 to 100 mg/mL. The four quinolone nucleoside
compounds (9–12) were all able to exert antiproliferative
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
Synthesis and Antiproliferative Activity of Quinolone Nucleosides
3
H
N
O
Br
O
O
1
1. BSA
2. Rib-Ac4 or Rib-Bz4
3. TMS-OTf
MW or
conventional
O
O
O
Br
only for 3
only for 2
Buchwald-Hartwig
coupling
RO
O
N
1. NaOMe/EtOH
R = Ac: 2
R = Bz: 3
cat. Pd(OAc)2,
BINAP,
2. TBDMSCl,
imidazol
OR
RO
,
Cs2CO3 aniline
O
N
O
O
O
O
O
4
H
N
Br
O
O
BzO
TBDMSO
N
OBz
BzO
OTBDMS
TBDMSO
O
O
O
5
X
O
Buchwald-Hartwig
coupling
TBDMSO
N
For 6: cat. Pd(OAc)2,
NH3/MeOH
X = O:
6
BP(tBu)2,
X = NH: 7
OTBDMS
TBDMSO
,
K3PO4 phenol
For 7: cat. Pd(OAc)2,
NEt3* 3 HF,
NEt3
BINAP,
,
Cs2CO3 aniline
O
N
O
O
O
O
H
N
X
H2N
HO
O
HO
HO
N
O
O
X = O:
8
X = NH: 9
10
OH
OH
only for 8
HO
O
N
O
O
O
O
O
NH3/
MeOH
O
LiOH
HO
HO
H2N
HO
HO
HO
N
11
12
OH
OH
Scheme 1. Synthesis strategies for 6-anilino-and 6-phenoxy-substituted quinolone nucleosides (9–12) as target compounds via Buchwald–
Hartwig cross-coupling as key step.
activity against K562 cells with IC50 values of 98.27 ꢀ 2.11,
64.09 ꢀ 2.14, 198.50 ꢀ 3.25, and 76.77 ꢀ 2.33 mM, respectively
(Table 1). Since compounds (10) and (12) are the most active
compounds of this list carrying a carboxamide modification,
it looks that the amide portion is preferred over ester and free
acid.
Table 1 also reports the effects of the employed compounds
on K562 erythroid differentiation. This activity was assayed
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

4
L. Wicke et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
Table 1. Effect of the four 6-substituted quinolone nucleosides
(9–12) on K562 cell growth and differentiation after 5 days of
culture at the indicated concentrations.
acid, or amide functions in good yields, ready for testing
biologically. Among the synthesized compounds, two were
found to exert efficient antiproliferative activity on the
human CML K562, and behave as antiproliferative agents
acting without induction of differentiated functions (in this
case erythroid differentiation). These novel molecules deserve
further experiments to determine their antiproliferative
effects on other CML cell lines.
Quinolone
nucleoside
Differentiation
max (%)
IC50 (mM)
9
10
11
12
98.27 ꢀ 2.11
64.09 ꢀ 2.14
198.50 ꢀ 3.25
76.77 ꢀ 2.33
250 ꢀ 7 (nM)
2 ꢀ 0.4 (100 mM)
4 ꢀ 0.8 (50 mM)
4 ꢀ 0.3 (200 mM)
3 ꢀ 0.6 (75 mM)
92 ꢀ 3 (500 nM)
AraC
Experimental
IC50 values represent the average ꢀ SD of three independent
experiments. Differences within and between groups were
evaluated by one-way analysis of variance test ꢂꢂꢂp < 0.0001
(F ¼ 187.6, r2 ¼ 0.98) followed by a multi-comparison Dunnett’s
test: ꢂꢂp < 0.01 compared with the positive control ara-C.
General
Chemicals and reagents used for the study of antiproliferative
activities were purchased from Sigma–Aldrich Co., while other
chemicals, solvents, and reagents were purchased from VWR
(Milan, Italy). The fetal bovine serum was obtained from CELBIO
(Milano, Italy). All reagents for chemical synthesis were of the
highest commercially available quality and were used as received.
For column chromatography, technical solvents were distilled
before use. Thin layer chromatograms (TLC) were recorded on
60 F254 plates from Merck (thickness of layer 0.2 mm). Column
chromatography was carried out on silica gel 60 (40–63 mm,
Merck). Reversed phase (RP) HPLC was performed on a Jasco
LC-2000Plus HPLC system equipped with a Jasco UV 2075Plus
detector (detection at 254 nm) and a preparative Phenomenex
Jupiter Proteo C12 column (250 mm ꢁ 15 mm), and the eluents
Millipore water and acetonitrile with a flow of 6 mL/min. NMR
spectra were recorded on Bruker AM, DPX, and AV instruments at
250, 300, and 400 MHz and 300 K. Chemical shifts (d) are reported
in ppm relative to the solvent signal. The fine structure of proton
signals was specified with s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), dd (doublet of doublet), dt (doublet of
triplet), and bs (broad singlet).
using a compound concentration causing 50% inhibition of
cell growth. None of the compounds were able to induce a
significant increase in the proportion of benzidine-positive
cells, an indication of the activation of the erythroid pathway.
This lack of effects on erythroid induction was further assayed
in experiments of co-treatment by adding the quinolone
nucleoside compounds (9–11) to suboptimal concentrations
(12 nM) of mithramycin (Table 2), a known inducer of
erythroid differentiation [29]. The obtained results indicated
no synergistic effects of the quinolone nucleoside compounds,
as they were not able to stimulate a significant increase in the
mithramycin-induced proportion of erythroid differentiation
of benzidine-positive K562 cells.
ESI-mass spectrometry was performed on a Fisons instrument
equipped with a VG platform II with quadrupol analyzer, and
MALDI mass spectra were recorded on a Fisons VG Tofspec
spectrometer. High-resolution mass spectrometry (HRMS) was
provided by a MALDI Orbitrap LTQ XL instrument (Thermo
Fisher). Microwave-assisted synthesis was carried out in an open
vessel using the MW lab instrument CEM Discover.
Conclusions
In conclusion, we developed a versatile synthesis approach to
prepare 6-substituted quinolone nucleosides bearing hetero-
atom-bridged phenol or aniline derivatives, and varying ester,
Chemical synthesis
Synthesis of 6-bromo-4-quinolone-3-ethylcarboxylate (1)
This synthesis was performed according to described proce-
dures [40, 41] and analytical data of this particular bromo
derivative were found to be in agreement with the ones
previously reported [50].
Table 2. Level of K562 differentiation induced by mithramycin in
the presence of quinolone nucleosides (9–12).
Inducer
Differentiation max (%)
9 þ mithr. 2
38 ꢀ 3.4 (100 mM) þ mithr (12 nM)
39 ꢀ 3.5 (65 mM) þ mithr (12 nM)
40 ꢀ 4.2 (200 mM) þ mithr (12 nM)
37 ꢀ 2.7 (75 mM) þ mithr (12 nM)
87 ꢀ 7.8 (25 nM)
Synthesis of 6-bromo-1-(20,30,50-tri-O-acetyl-b-D-
ribofuranosyl)-4-quinolone-3-ethyl-carboxylate (2)
10 þ mithr. 2
11 þ mithr. 2
12 þ mithr. 2
Mithramycin 1
Mithramycin 2
Conventional heating: In a dry flask under argon, 1.5 g
(5.07 mmol) 6-bromo-4-quinolone-3-ethylcarboxylate 1 and 1.94 g
10,20,30,50-O-tetraacetyl-b-D-ribofuranose
35 ꢀ 3.4 (12 nM)
(6.08 mmol,
1.2 eq.)
were suspended in 42 mL abs. acetonitrile. 1.49 mL (6.08 mmol,
1.2 eq.) N,O-bis(trimethylsilyl)acetamide (BSA) was added and the
cloudy mixture heated under reflux at 80°C for 30 min.
Afterwards, the clear solution was cooled to room temperature,
1.1 mL (6.08 mmol, 1.2 eq.) trimethylsilyl-trifluormethanesulfo-
nate (TMS-OTf) was added, and the reaction mixture again
Differentiation max (%) values represent the average ꢀ SD of three
independent experiments. Differences within and between
groups were evaluated by one way analysis of variance test
ꢂꢂꢂp < 0.0001 (F ¼ 187.6, r2 ¼ 0.98) followed by a multi-compari-
son Dunnett’s test: ꢂꢂp < 0.01 compared with mithramycin 1.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
Synthesis and Antiproliferative Activity of Quinolone Nucleosides
5
heated to 80°C for 3 h. The reaction solution was allowed to cool
down and was stopped with aqueous saturated NaHCO3.
Extraction was performed with DCM for several times, and the
combined organic layers were dried over MgSO4 and concentrated
in vacuo. Further purification was carried out by column
chromatography (eluent 95:5 DCM/MeOH), and the pure product
2 could be obtained as off-white foam in 2.8 g (5.05 mmol, 99%)
yield.
MALDI(þ)-MS (m/z) calcd.: 739.11 [C38H30BrNO10], found: 741.8
[MþH]þ.
Synthesis of 6-bromo-1-(b-D-ribofuranosyl)-4-quinolone-3-
ethyl-carboxylate (4a)
One gram (1.8 mmol) acetyl-protected compound 2 was dissolved
in 20 mL abs. ethanol and 54 mg (1.08 mmol, 0.6 eq.) sodium
methoxide was added. The yellow solution was stirred for 90 min
at room temperature (reaction control by TLC, 9:1 DCM/MeOH),
while a precipitate formed. After the reaction was finished, the pH
was adjusted to 7 by addition of a spatula of Dowex 50 W X 8.
Subsequently, the reaction mixture was filtrated, the residue
washed with methanol, and the filtrate concentrated to dryness.
The crude product was purified by column chromatography
(eluent 9:1 DCM/MeOH) to give a light-yellow solid in 0.75 g
(1.75 mmol, 97%) yield.
Microwave-induced heating: The setup was identical to the
one for conventional heating in an open 100 mL flask under
argon and reflux condenser: 1.8 g (6.08 mmol) 6-bromo-4-quino-
lone-3-ethylcarboxylate 1 and 2.32 g (7.3 mmol, 1.2 eq.) 10,20,30,50-
O-tetraacetyl-b-D-ribofuranose were suspended in 50 mL abs.
acetonitrile; the MW irradiation time was 10 min at 80°C and
100 W. After addition of 1.3 mL (7.3 mmol, 1.2 eq.) trimethylsilyl-
trifluormethanesulfonate, the reaction mixture was heated via
MW irradiation at 80°C and 100 W for 60 min and subsequently
quenched with saturated NaHCO3 solution. Work-up and
purification were performed in the same way as described for
the conventional method to give 3.3 g (5.95 mmol, 98%) product 2
as light-yellow foam.
TLC: Rf ¼ 0.2 (9:1 DCM/MeOH). 1H NMR: d [ppm] (250 MHz,
DMSO-d6): 9.11 (s, 1H, H2); 8.24 (d, J ¼ 2.5 Hz, 1H, H5); 7.89
(dd, J ¼ 9.1 Hz, J ¼ 2.5 Hz, 1H, H7); 7.73 (d, J ¼ 9.2 Hz, 1H, H8); 5.98
(d, J ¼ 3.2 Hz, 1H, H10); 5.75 (d, J ¼ 5.4 Hz, 1H, 20OH); 5.22
(d, J ¼ 5.8 Hz, 1H, 30OH); 5.08 (dd, J ¼ 4.8 Hz, J ¼ 4.7 Hz, 1H,
50OH); 4.16 (m, 5H, H20, H30, H40, H50, H500); 3.69 (m, 2H, CH2);
1.19 (dd, J ¼ 7.1 Hz, J ¼ 7.0 Hz, 3H, CH3). 13C NMR: d [ppm]
TLC: Rf ¼ 0.36 (95:5 DCM/MeOH). 1H NMR: d [ppm] (250 MHz,
DMSO-d6): 8.75 (s, 1H, H2); 8.24 (d, J ¼ 2.5 Hz, 1H, H5); 7.89
(dd, J¼ 9.1 Hz, J ¼ 2.5 Hz, 1H, H7); 7.70 (d, J ¼ 9.3 Hz, 1H, H8); 6.50
(d, J ¼ 4.9 Hz, 1H, H10); 5.49 (dd, J ¼ 5.0 Hz, J ¼ 5.9 Hz, 1H, H20); 5.31
(dd, J ¼ 5.9 Hz, J ¼ 5.4 Hz, 1H, H30); 4.47 (m, 1H, H40); 4.33
(d, J ¼ 3.4 Hz, 2H, H50, H500); 4.16 (m, 2H, CH2); 1.20 (dd, J ¼ 7.1 Hz,
J ¼ 7.0 Hz, 3H, CH3). 13C NMR: d [ppm] (62.9 MHz, CDCl3): 172.91
–
–
–
(62.9 MHz, DMSO-d ): 171.77 (C O quinolone); 166.95 (C O ester),
–
6
140.55 (C2); 137.60 (C9); 131.67 (C7); 129.26 (C5); 128.44 (C10);
123.28 (C8); 118.03 (C–Br); 110.61 (C3); 92.28 (C10); 85.04 (C40);
78.84 (C20); 69.13 (C30); 67.38 (C50); 58.80 (CH2 ester); 14.16 (CH3
ester). ESI(ꢃ)-MS (m/z) calcd.: 427.03 [C17H18BrNO7], found: 428.0
[MꢃH]ꢃ.
–
–
–
–
(C O quinolone); 170.43 (C O acetyl); 169.38 (C O acetyl); 168.85
–
–
Synthesis of 6-bromo-1-(20,30,50-tri-O-tert-
butyldimethylsilyl-b-D-ribofuranosyl)-4-quinolone-3-ethyl-
carboxylate (4)
–
–
(C O acetyl); 165.14 (C O ester), 143.15 (C2); 137.47 (C9); 135.61
–
–
(C7); 130.90 (C5); 130.10 (C10); 119.47 (C8); 116.42 (C–Br); 112.67
(C3); 89.12 (C10); 81.04 (C40); 73.97 (C20); 70.21 (C30); 62.62 (C50);
61.21 (CH2 ester); 20.68 (CH3 acetyl); 20.53 (CH3 acetyl); 20.34
(CH3 acetyl); 14.39 (CH3 ester). ESI(þ)-MS (m/z) calcd.: 553.06
[C23H24BrNO10], found: 554.1 [MþH]þ.
To a solution of 0.88 g (5.84 mmol, 10 eq.) tert-butyldimethylsilyl
chloride (TBDMSCl) and 0.8 g (11.67 mmol, 20 eq.) imidazole in
5 mL abs. DMF, unprotected nucleoside 4a was added under
argon. The reaction mixture was stirred for 42 h at room
temperature (reaction control by TLC, 95:5 DCM/MeOH). Subse-
quently, saturated NaHCO3 solution was added and the mixture
was extracted with DCM. The combined organic phases were
washed with water, dried over MgSO4, and evaporated. The
residue was purified via column chromatography (eluent 98:2
DCM/MeOH) to yield 0.43 g (0.56 mmol, 96%) of a light-yellow solid
as pure product 4.
Synthesis of 6-bromo-1-(20,30,50-tri-O-benzoyl-b-D-
ribofuranosyl)-4-quinolone-3-ethyl-carboxylate (3)
Vorbrüggen glycosylation to synthesize compound
3 was
performed via conventional heating, and identical conditions
were described for preparation of 2. The difference was the
use of 5 g (9.91 mmol, 1.2 eq.) 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-
ribofuranose for 2.45 g (8.26 mmol) 6-bromo-4-quinolone-3-ethyl-
carboxylate 1 in 85 mL abs. acetonitrile and 1.8 mL (9.91 mmol,
1.2 eq.) trimethylsilyl-trifluormethanesulfonate in the second
step. Also work-up and purification were identical and yielded
6.05 g (8.2 mmol, 99%) off-white foam as product 3.
TLC: Rf ¼ 0.73 (95:5 DCM/MeOH). 1H NMR: d [ppm] (250 MHz,
CDCl3): 8.83 (s, 1H, H2); 8.62 (m, 1H, H5); 7.69 (m, 2H, H7, H8); 6.13
(d, J ¼ 7.5 Hz, 1H, H10); 4.37 (m, 3H, H20, H30, H40); 4.20 (m, 2H,
H50, H500); 3.89 (m, 2H, CH2); 1.40 (dd, J ¼ 7.1 Hz, J ¼ 7.0 Hz, 3H,
CH3); 0.97 (s, 9H, TBDMS: tBu); 0.94 (s, 9H, TBDMS: tBu); 0.72 (s, 9H,
TLC: Rf ¼ 0.23 (95:5 DCM/MeOH). 1H NMR: d [ppm] (250 MHz,
CDCl3): 8.89 (s, 1H, H2); 8.57 (d, J ¼ 2.1 Hz, 1H, H5); 7.95 (m, 8H, H7,
H8, 6H of Bz); 7.43 (m, 9H, Bz); 6.39 (d, J ¼ 5.1 Hz, 1H, H10); 5.93
(dd, J ¼ 5.4 Hz, J ¼ 5.5 Hz, 1H, H20); 5.84 (dd, J ¼ 5.6 Hz, J ¼ 4.5 Hz,
1H, H30); 4.83 (m, 3H, H40, H50, H500); 4.10 (m, 2H, CH2); 1.18
(dd, J ¼ 7.1 Hz, J ¼ 7.0 Hz, 3H, CH3); 13C NMR: d [ppm] (62.9 MHz,
t
TBDMS: Bu); 0.17 (s, 3H, TBDMS: methyl); 0.15 (s, 3H, TBDMS:
methyl); 0.13 (s, 3H, TBDMS: methyl); 0.10 (s, 3H, TBDMS: methyl);
ꢃ0.08 (s, 3H, TBDMS: methyl); ꢃ0.46 (s, 3H, TBDMS: methyl).
13
–
C NMR: d [ppm] (62.9 MHz, CDCl ): 172.13 (C O quinolone);
–
3
–
164.33 (C O ester), 143.05 (C2); 137.43 (C9); 134.25 (C7); 129.29
–
–
–
–
–
CDCl ): 172.04 (C O quinolone); 165.01 (C O Bz); 164.11 (C O Bz);
(C5); 128.97 (C10); 117.95 (C8); 111.17 (C–Br); 111.43 (C3); 89.33
(C10); 86.48 (C40); 77.90 (C20); 72.08 (C30); 62.23 (C50); 59.95 (CH2
ester); 25.09 (Si–C–(CH3)3); 24.82 (Si–C–(CH3)3); 24.67 (Si–C–(CH3)3);
17.50 (Si–C–(CH3)3); 17.04 (Si–C–(CH3)3); 16.72 (Si–C–(CH3)3); 13.43
(CH3 ester); ꢃ5.35 (Si–CH3); ꢃ5.53 (Si–CH3); ꢃ5.61 (Si–CH3); ꢃ6.38
(Si–CH3); ꢃ6.40 (Si–CH3); ꢃ6.68 (Si–CH3); MALDI(þ)-MS (m/z) calcd.:
769.3 [C35H60BrNO7Si3], found: 772.7 [MþH]þ.
–
–
3
–
–
163.66 (C O Bz); 163.31 (C O ester), 137.67 (C2); 136.29 (C9);
–
–
135.71 (C7); 134.64 (C5); 133.17 (Bz); 132.95 (Bz); 132.67 (Bz);
130.63 (Bz); 129.92 (Bz); 129.84 (Bz); 129.16 (Bz); 128.81 (Bz); 128.70
(Bz); 127.44 (Bz); 127.71 (Bz); 127.61 (Bz); 127.32 (C10); 126.95 (C8);
115.56 (C–Br); 111.50 (C3); 89.47 (C10); 80.17 (C40); 73.27 (C20);
69.84 (C30); 62.38 (C50); 59.93 (CH2 ester); 13.20 (CH3 ester).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
L. Wicke et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
Buchwald–Hartwig cross-coupling
General procedure for coupling with phenol
tri-O-benzoyl-b-D-ribofuranosyl)-4-quinolone-3-ethyl-carboxylate 3,
6 mg (0.027mmol, 0.1 eq.) Pd(OAc)2, 25 mg (0.0405 mmol,
0.15eq.) (ꢀ)-BINAP, and 0.1 g (0.324 mmol, 1.4 eq.) cesium carbon-
ate as well as 0.030 mL (0.324 mmol, 1.4 eq.) aniline in 3 mL toluene
were used. The reaction was finished after 18 h at 90°C (reaction
control via TLC: DCM/MeOH 95:5) and the product was purified as
described above (column chromatography with eluent DCM/MeOH
95:5). The pure product 5 could be obtained as light-yellow solid in
96% (0.19 g, 0.258 mmol) yield.
Dry silyl-protected bromo nucleoside, 10 mol% palladium(II)
acetate [Pd(OAc)2], and 15 mol% (2-biphenyl)-di-tert-butylphosphin
[BP(tBu)2] were suspended in abs. toluene under argon atmo-
sphere and stirred for 5 min. Subsequently, 2 eq. potassium
phosphate and 1.2 eq. phenol were added. The reaction mixture
was heated under reflux at 100°C for typically 18 h. After
evaporation of the solvent, the residue was purified by column
chromatography (eluent typically hexane/ethyl acetate 7:3) and
the pure product dried under high vacuum.
TLC: Rf ¼ 0.16 (DCM/MeOH 95:5). 1H NMR: d [ppm] (250 MHz,
CDCl3): 8.83 (s, 1H, H2); 8.73 (bs, 1H, NH); 8.01–6.84 (m, 18H, H5,
H7, H8, 15H benzoyl); 6.63 (m, 5H, aniline); 6.38 (d, J ¼ 4.8 Hz, 1H,
H10); 5.94 (dd, J ¼ 5.5 Hz, J ¼ 4.9 Hz, 1H, H20); 5.83 (dd, J ¼ 5.1 Hz,
J ¼ 5.5 Hz, 1H, H30); 4.77 (m, 3H, H40, H50, H500); 4.06 (m, 2H, CH2);
1.17 (dd, J ¼ 7.1 Hz, J ¼ 7.0 Hz, 3H, CH3). 13C NMR: d [ppm]
6-Phenoxy-1-(20,30,50-tri-O-tert-butyldimethylsilyl-b-D-ribo-
furanosyl)-4-quinolone-3-ethyl-carboxylate (6): According
to the general procedure described above, 0.27 g (0.35 mmol) 6-
bromo-1-(20,30,50-tri-O-tert-butyldimethylsilyl-b-D-ribofuranosyl)-4-
quinolone-3-ethyl-carboxylate 4, 8 mg (0.035 mmol, 0.1 eq.) Pd(OAc)2,
16 mg (0.053 mmol, 0.15 eq.) BP(tBu)2, and 0.15 g (0.7 mmol, 2 eq.)
potassium phosphate as well as 0.04 g (0.42 mmol, 1.2 eq.) phenol in
5.5 mL toluene were employed. The reaction was finished after 16 h
(reaction control via TLC: DCM/MeOH 95:5) and the product was
purified as described above. The pure product 6 could be obtained as
light-yellow, crystalline solid in 81% (0.22 g, 0.28 mmol) yield.
TLC: Rf ¼ 0.41 (7:3 hexane/EE). 1H NMR: d [ppm] (250 MHz,
CDCl3): 8.83 (s, 1H, H2); 8.07 (d, J ¼ 2.9 Hz, 1H, H5); 7.83
(d, J ¼ 9.5 Hz, 1H, H8); 7.34 (m, 2H, H7, phenoxy); 7.16 (m, 2H,
phenoxy); 7.01 (m, 2H, phenoxy); 6.18 (d, J ¼ 7.7 Hz, 1H, H10); 4.37
(m, 3H, H20, H30, H40); 4.20 (m, 2H, H50, H500); 3.90 (m, 2H, CH2);
–
–
–
(62.9 MHz, CDCl ): 174.07 (C O quinolone); 166.10 (C O bz);
–
3
–
–
–
165.14 (C O bz); 164.72 (C O bz); 162.42 (C O ester), 146.39 (C2);
–
–
–
141.84 (aniline); 137.84 (C9); 134.57 (bz); 129.83 (bz); 129.52
(aniline); 129.31 (bz); 128.74 (C6); 128.61 (C 10); 126.48 (C7); 118.78
(aniline); 118.60 (aniline); 116.82 (C5); 115.14 (C8); 110.02 (C3);
89.30 (C10); 80.37 (C40); 75.23 (C20); 69.12 (C30); 59.60 (C50); 58.90
(CH2 ester); 14.30 (CH3 ester). MALDI(þ)-MS (m/z) calcd.: 752.24
[C44H36N2O10], found: 754.5 [MþH]þ.
6-Phenylamino-1-(20,30,50-tri-O-tert-butyldimethylsilyl-b-D-
ribofuranosyl)-4-quinolone-3-ethyl-carboxylate (7): Accord-
ing to the general procedure described above, 0.15 g (0.19 mmol)
6-bromo-1-(20,30,50-tri-O-tert-butyldimethylsilyl-b-D-ribofuranosyl)-
4-quinolone-3-ethyl-carboxylate 4, 4.3 mg (0.027 mmol, 0.1 eq.)
Pd(OAc)2, 18 mg (0.029 mmol, 0.15 eq.) (ꢀ)-BINAP, and 88 mg
(0.27 mmol, 1.4 eq.) cesium carbonate as well as 0.025 mL
(0.27 mmol, 1.4 eq.) aniline in 4 mL toluene were employed.
The reaction was finished after 17.5 h at 85°C (reaction control
via TLC: DCM/MeOH 95:5) and the product was purified as
described above (column chromatography with eluent hexane/
ethyl acetate 7:3). The pure product 7 could be obtained as light-
yellow foam in 98% (0.15 g, 0.18 mmol) yield.
t
1.39 (dd, J ¼ 7.1 Hz, J ¼ 7.0 Hz, 3H, CH3); 0.97 (s, 9H, TBDMS: Bu);
t
t
0.94 (s, 9H, TBDMS: Bu); 0.72 (s, 9H, TBDMS: Bu); 0.17 (s, 3H,
TBDMS: methyl); 0.15 (s, 3H, TBDMS: methyl); 0.13 (s, 3H, TBDMS:
methyl); 0.10 (s, 3H, TBDMS: methyl); ꢃ0.07 (s, 3H, TBDMS:
methyl); ꢃ0.44 (s, 3H, TBDMS: methyl). 13C NMR: d [ppm]
–
–
–
(62.9 MHz, CDCl ): 172.82 (C O quinolone); 164.82 (C O ester),
–
3
155.87 (phenoxy); 153.86 (C2); 134.30 (C6); 129.18 (C9); 128.93
(phenoxy); 128.53 (C7); 123.11 (phenoxy); 122.93 (C10); 119.93
(C5); 118.22 (phenoxy); 114.36 (C8); 110.27 (C3); 86.38 (C10); 76.19
(C40); 75.73 (C20); 72.10 (C30); 62.26 (C50); 59.81 (CH2 ester); 25.08
(Si–C–(CH3)3); 24.83 (Si–C–(CH3)3); 24.69 (Si–C–(CH3)3); 17.49
(Si–C–(CH3)3); 17.04 (Si–C–(CH3)3); 16.75 (Si–C–(CH3)3); 13.43
(CH3 ester); ꢃ5.34 (Si–CH3); ꢃ5.53 (Si–CH3); ꢃ5.59 (Si–CH3);
ꢃ6.41 (Si–CH3); ꢃ6.43 (Si–CH3); ꢃ6.68 (Si–CH3). MALDI(þ)-MS (m/z)
calcd.: 783.40 [C41H65NO8Si3], found: 785.59 [MþH]þ.
TLC: Rf ¼ 0.12 (7:3 hexane/EE). 1H NMR: d [ppm] (250 MHz,
CDCl3): 8.62 (s, 1H, H2); 7.99 (d, J ¼ 2.7 Hz, 1H, H5); 7.58 (d,
J ¼ 9.3 Hz, 1H, H8); 7.26 (dd, J ¼ 9.2 Hz, J ¼ 2.8 Hz, 1H, H7); 7.13 (m,
2H, aniline meta); 6.98 (m, 2H, aniline ortho); 6.82 (m, 1H, aniline
para); 6.01 (d, J ¼ 7.7 Hz, 1H, H10); 5.97 (bs, 1H, NH); 4.22 (m, 3H,
H20, H30, H40); 4.04 (m, 2H, H50, H500); 3.84 (m, 2H, CH2); 1.25 (dd,
J ¼ 7.1 Hz, J ¼ 7.0 Hz, 3H, CH3); 0.83 (s, 9H, TBDMS: tBu); 0.79 (s, 9H,
TBDMS: tBu); 0.59 (s, 9H, TBDMS: tBu); 0.02 (s, 3H, TBDMS: methyl);
0.00 (s, 3H, TBDMS: methyl); ꢃ0.02 (s, 3H, TBDMS: methyl); 0.08 (s,
3H, TBDMS: methyl); ꢃ0.23 (s, 3H, TBDMS: methyl); ꢃ0.58 (s, 3H,
General procedure for coupling with aniline
Dry benzoyl- or silyl-protected bromo nucleoside, 10 mol%
palladium(II) acetate [Pd(OAc)2] and 15 mol% (ꢀ)-2,20-bis-
(diphenylphosphino)-1,10-binaphthaline [(ꢀ)-BINAP] were sus-
pended in abs. toluene under argon atmosphere, and afterwards,
1.4 eq. cesium carbonate and 1.4 eq. aniline were added. The
reaction mixture was stirred at room temperature for 30 min
under exclusion of light and subsequently heated under reflux at
typically 90°C for typically 18 h. After cooling down, ethyl acetate
was added and the whole mixture centrifuged (4000 rpm,
10 min). The supernatant was concentrated in vacuo, the residue
purified by column chromatography, and the pure product dried
under high vacuum.
TBDMS: methyl). 13C NMR: d [ppm] (62.9 MHz, CDCl ): 173.06 (C O
–
–
3
–
quinolone); 164.97 (C O ester), 141.30 (C2); 139.90 (aniline);
–
132.57 (C9); 129.03 (C6); 128.42 (aniline); 121.45 (C7); 120.75 (C10);
117.30 (aniline); 116.50 (C5); 112.33 (C8); 109.73 (C3); 86.21 (C10);
76.22 (C40); 75.65 (C20); 72.07 (C30); 62.28 (C50); 59.67 (CH2 ester);
24.73 (Si–C–(CH3)3); 24.84 (Si–C–(CH3)3); 25.08 (Si–C–(CH3)3); 17.49
(Si–C–(CH3)3); 17.04 (Si–C–(CH3)3); 16.76 (Si–C–(CH3)3); 13.49 (CH3
ester); ꢃ5.36 (Si–CH3); ꢃ5.53 (Si–CH3); ꢃ5.62 (Si–CH3); ꢃ6.39
(Si–CH3); ꢃ6.40 (Si–CH3); ꢃ6.66 (Si–CH3). ESI(þ)-MS (m/z) calcd.:
781.41 [C41H65N2O7Si3], found: 783.6 [MþH]þ.
6-Phenylamino-1-(20,30,50-tri-O-benzoyl-b-D-ribofuranosyl)-
General procedure for silyl deprotection
Dry silyl-protected 6-substituted quinolone nucleosides were
dissolved in THF, and at first 2 eq. triethylamine and afterwards
4-quinolone-3-ethyl-carboxylate (5): According to the general
procedure described above, 0.20 g (0.27 mmol) 6-bromo-1-(20,30,50-
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
Synthesis and Antiproliferative Activity of Quinolone Nucleosides
7
4.5 eq. triethylamine-trihydrofluoride were added under argon.
The reaction solution was stirred at room temperature typically
overnight. The solvent was evaporated and the residue purified by
column chromatography (eluent typically 9:1 DCM/MeOH). The
pure product was dried under high vacuum.
saturated with ammonia at ꢃ10°C (NH3/MeOH) under argon and
stirred for typically 3 days at room temperature. The solvent was
evaporated, the residue washed with chloroform and purified
by column chromatography (eluent 9:1 DCM/MeOH) or RP-HPLC
(25–38% acetonitrile in water in 0–20 min). The pure product was
dried under high vacuum.
6-Phenoxy-1-(b-D-ribofuranosyl)-4-quinolone-3-ethyl-car-
6-Phenylamino-1-(b-D-ribofuranosyl)-4-quinolone-3-carb-
boxylate (8): According to the general procedure described
above, 0.064 g (0.082 mmol) 6-phenoxy-1-(20,30,50-tri-O-tert-butyldi-
methylsilyl-b-D-ribofuranosyl)-4-quinolone-3-ethyl-carboxylate 6,
60 mL (0.37 mmol, 4.5 eq.) triethylamine-trihydrofluoride, and
23 mL (0.164 mmol, 2 eq.) triethylamine in 1.5 mL abs. THF were
employed. The reaction was finished after 16 h (reaction control
via TLC: DCM/MeOH 95:5) and the product was purified as
described above. The pure product 8 could be obtained as
colorless crystalline solid in 66% (26 mg, 0.054 mmol) yield.
TLC: Rf ¼ 0.24 (DCM/MeOH 9:1). 1H NMR: d [ppm] (400 MHz,
DMSO-d6): 9.14 (s, 1H, H2); 7.92 (d, J ¼ 9.3 Hz, 1H, H8); 7.63
(d, J ¼ 2.9 Hz, 1H, H5); 7.57 (dd, J ¼ 9.2 Hz, J ¼ 3.0 Hz, 1H, H7); 7.46
(m, 2H, phenoxy); 7.24 (m, 1H, phenoxy); 7.20 (m, 2H, phenoxy);
6.08 (d, J ¼ 3.2 Hz, 1H, H10); 5.81 (d, J ¼ 5.4 Hz, 1H, 20OH); 5.26
(d, J ¼ 5.9 Hz, 1H, 30OH); 5.16 (t, J ¼ 4.7 Hz, 1H, 50OH); 4.18 (m, 3H,
H20, H30, H40); 4.06 (m, 2H, CH2); 3.75 (m, 2H, H50, H500); 1.26
(t, J ¼ 7.1 Hz, 3H, CH3). 13C NMR: d [ppm] (62.9 MHz, DMSO-d6):
oxy-amide (10): According to the general procedure described
above, 0.17 g (0.23 mmol) 6-phenylamino-1-(20,30,50-tri-O-benzoyl-
b-D-ribofuranosyl)-4-quinolone-3-ethyl-carboxylate 5 was dissolved
in 10 mL NH3/MeOH and stirred for 4 days (reaction control via
TLC DCM/MeOH 9:1). After purification using column chroma-
tography as described above, the pure product 10 was obtained as
yellow crystalline solid in 59% (56 mg, 0.136 mmol) yield.
TLC: Rf ¼ 0.13 (DCM/MeOH 9:1). 1H NMR: d [ppm] (400 MHz,
DMSO-d6): 9.36 (d, J ¼ 4.5 Hz, 1H, NH amide, exchange of 40% with
D2O); 9.13 (s, 1H, H2); 8.67 (bs, 1H, NH aniline, H/D-exchange with
D2O); 8.05 (d, J ¼ 3.0 Hz, 1H, H5); 7.92 (d, J ¼ 9.6 Hz, 1H, H8); 7.58
(dd, J ¼ 9.3 Hz, J ¼ 2.9 Hz, 1H, H7); 7.46 (d, 1H, J ¼ 4.5 Hz, 1H, NH
amide, exchange of 40% with D2O); 7.38 (m, 2H, aniline meta), 7.24
(m, 2H, aniline ortho); 7.00 (m, 1H, aniline para), 6.13 (d, J ¼ 4.1 Hz,
1H, H10); 5.81 (d, J ¼ 5.9 Hz, 1H, 20OH); 5.37 (d, J ¼ 5.8 Hz, 1H,
30OH); 5.15 (t, J ¼ 5.0 Hz, 1H, 50OH); 4.27 (m, 1H, H20); 4.17 (m, 1H,
H40); 4.04 (m, 1H, H30); 3.77 (m, 2H, H50, H500). 13C NMR: d [ppm]
–
–
–
172.27 (C O quinolone); 164.28 (C O ester); 155.89 (phenoxy);
–
–
(100.6 MHz, DMSO-d ): 175.04 (C O quinolone); 166.95 (C O
–
6
–
154.47 (C2); 134.35 (C6); 130.29 (phenoxy); 129.22 (C9); 127.81
(C7); 125.05 (phenoxy); 124.31 (C10); 123.99 (C5); 119.39
(phenoxy); 109.45 (C8); 103.94 (C3); 92.35 (C10); 85.07 (C40);
83.68 (C20); 74.81 (C30); 69.13 (C50); 59.64 (CH2 ester); 13.43 (CH3
ester). MALDI(þ)-MS (m/z) calcd.: 441.14 [C23H23NO8], found:
442.79 [MþH]þ.
–
ester); 142.42 (C2); 141.47 (aniline); 132.26 (C9); 131.69 (C6);
129.34 (aniline); 128.64 (C7); 128.33 (C10); 120.88 (C5); 118.38
(aniline); 117.66 (aniline); 111.19 (C8); 108.69 (C3); 85.31 (C10);
74.36 (C40); 69.74 (C20); 67.38 (C30); 60.96 (C50). MALDI(þ)-MS (m/z)
calcd.: 411.14 [C21H21N3O6], found: 413.32 [MþH]þ. HRMS (MþH):
calcd.: 412.15031, found: 412.15139.
6-Phenylamino-1-(b-D-ribofuranosyl)-4-quinolone-3-ethyl-
6-Phenoxy-1-(b-D-ribofuranosyl)-4-quinolone-3-carboxy-
carboxylate (9): According to the general procedure described
above, 0.070 g (0.089 mmol) 6-phenylamino-1-(20,30,50-tri-O-tert-
butyldimethylsilyl-b-D-ribofuranosyl)-4-quinolone-3-ethyl-carbox-
ylate 7, 66 mL (0.402 mmol, 4.5 eq.) triethylamine-trihydrofluoride,
and 25 mL (0.179 mmol, 2 eq.) triethylamine in 1.5mL abs. THF
were employed. The reaction was finished after 17 h (reaction
control via TLC: DCM/MeOH 95:5) and the product was purified as
described above. The pure product 8 could be obtained as pale-
yellow crystalline solid in 76% (30 mg, 0.068 mmol) yield.
amide (12): According to the general procedure described
above, 39 mg (0.088 mmol) 6-phenoxy-1-(b-D-ribofuranosyl)-4-quin-
olone-3-ethyl-carboxylate 8 was dissolved in 5 mL NH3/MeOH and
stirred for 6 days. After purification as described above using RP-
HPLC, the pure product 12 was obtained as yellow crystalline
solid in 65% (24 mg, 0.057 mmol) yield.
HPLC: retention time: 18.75 min. 1H NMR: d [ppm] (250 MHz,
DMSO-d6): 9.17 (s, 1H, H2); 9.12 (d, J ¼ 4.0 Hz, 1H, NH amide); 8.03
(d, J ¼ 9.5 Hz, 1H, H8); 7.70 (d, J ¼ 2.9 Hz, 1H, H5); 7.63 (dd,
J ¼ 9.3 Hz, J ¼ 2.9 Hz, 1H, H7); 7.48 (m, 3H, NH amide and phenoxy
meta), 7.25 (m, 1H, phenoxy para); 7.14 (m, 2H, phenoxy ortho), 6.12
(d, J ¼ 4.1, 1H, H10); 5.79 (d, J ¼ 5.5 Hz, 1H, 20OH); 5.35 (d, J ¼ 5.1 Hz,
1H, 30OH); 5.10 (t, J ¼ 4.9 Hz, 1H, 50OH); 4.23 (m, 1H, H20); 4.10 (m,
1H, H40); 3.98 (m, 1H, H30); 3.71 (m, 2H, H50, H500). 13C NMR: d
TLC: Rf ¼ 0.22 (DCM/MeOH 9:1). 1H NMR: d [ppm] (250 MHz,
DMSO-d6): 9.04 (s, 1H, H2); 8.57 (bs, 1H, NH); 7.92 (d, J ¼ 2.7 Hz, 1H,
H5); 7.75 (d, J ¼ 9.5 Hz, 1H, H8); 7.48 (dd, J ¼ 9.3 Hz, J ¼ 2.9 Hz, 1H,
H7); 7.31 (m, 2H, aniline meta), 7.16 (m, 2H, aniline ortho); 6.92 (m,
1H, aniline para), 6.03 (d, J ¼ 3.0 Hz, 1H, H10); 5.78 (d, J ¼ 5.4 Hz,
1H, 20OH); 5.25 (d, J ¼ 5.6 Hz, 1H, 30OH); 5.14 (t, J ¼ 4.8 Hz, 1H,
50OH); 4.12 (m, 5H, H20, H30, H40, CH2); 3.77 (m, 2H, H50, H500);
1.27 (t, J ¼ 7.1 Hz, 3H, CH3). 13C NMR: d [ppm] (61.9 MHz, DMSO-d6):
–
–
[ppm] (75.4 MHz, DMSO-d ): 175.88 (C O quinolone); 165.26 (C O
–
–
6
ester); 155.82 (phenoxy); 150.86 (C2); 148.03 (C6); 143.12 (C9);
130.30 (phenoxy); 128.44 (C7); 124.36 (phenoxy); 123.06 (C5);
120.48 (C10); 119.47 (phenoxy); 110.82 (C8); 103.43 (C3); 82.70
(C10); 77.90 (C40); 64.95 (C20); 63.19 (C30); 57.97 (C50). MALDI(þ)-MS
(m/z) calcd.: 412.13 [C21H21N3O6], found: 414.65 [Mþ2H]þ.HRMS
(MþH): calcd.: 413.1349, found: 413.1345.
–
–
–
172.54 (C O quinolone); 166.95 (C O ester); 142.52 (C2); 141.26
–
(aniline); 131.74 (C9); 129.30 (C6); 128.63 (aniline); 126.30 (C7);
124.04 (C10); 120.81 (C5); 117.46 (aniline); 108.77 (C8); 103.37 (C3);
91.86 (C10); 86.60 (C40); 81.62 (C20); 74.74 (C30); 67.39 (C50); 59.47
(CH2 ester); 13.85 (CH3 ester). ESI(þ)-MS (m/z) calcd.: 440.16
[C23H24N2O7], found: 441.2 [MþH]þ. HRMS (MþH): calcd.:
441.16563, found: 441.16713.
Synthesis of 6-phenoxy-1-(b-D-ribofuranosyl)-4-quinolone-
3-carboxylic-acid (11): Twenty-four milligrams (0.0544 mmol)
6-phenoxy-1-(b-D-ribofuranosyl)-4-quinolone-3-ethyl-carboxylate 8
was dissolved in a mixture of 1.5 mL THF/1.5 mL H2O and 5 mg
(0.109 mmol, 2 eq.) lithiumhydroxide monohydrate was added.
General procedure for aminolysis
Dry unprotected or benzoyl-protected 6-substituted quinolone
nucleoside with ester function was dissolved in abs. methanol
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
L. Wicke et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
The reaction solution was stirred at room temperature for 18 h
(reaction control via TLC DCM/MeOH 9:1), the organic solvent
removed under vacuum, and the remaining aqueous solution
acidified to pH 3 with 2 M hydrochloric acid. During this
procedure, a white powder precipitated, which was filtrated,
washed with cold water, and dried under high vacuum to give
18 mg (0.044 mmol, 81%) pure product 11.
The authors have declared no conflict of interest.
References
[1] J. Sessions, Am. J. Health Syst. Pharm. 2007, 64, 4–9.
[2] B. J. Druker, Blood 2008, 112, 4808–4817.
TLC: Rf ¼ 0.38 (DCM/MeOH 9:1). 1H NMR: d [ppm] (250 MHz,
DMSO-d6): 14.99 (s, 1H, COOH); 9.45 (s, 1H, H2); 8.11 (d, J ¼ 9.6 Hz,
1H, H8); 7.77 (dd, J ¼ 9.6 Hz, J ¼ 3.0 Hz, 1H, H7); 7.68 (d, J ¼ 3.0 Hz,
1H, H5); 7.50 (m, 2H, phenoxy meta), 7.28 (m, 1H, phenoxy para);
7.17 (m, 2H, phenoxy ortho), 6.25 (d, J ¼ 2.7 Hz, 1H, H10); 5.89
(d, J ¼ 5.4 Hz, 1H, 20OH); 5.30 (d, J ¼ 6.0 Hz, 1H, 30OH); 5.24
[3] S. Aoki, D. Kong, K. Matsui, M. Kobayashi, Anticancer Drugs
2004, 15, 363–369.
[4] A. Szulawska, J. Arkusinska, M. Czyz, Biochem. Pharmacol.
2007, 73, 175–184.
[5] J. Jakubowska, M. Wasowska-Lukawska, M. Czyz, Eur. J.
Pharmacol. 2008, 596, 41–49.
(t, J ¼ 4.3 Hz, 1H, 50OH); 4.13 (m, 3H, H20, H30, H40); 3.78 (m, 2H,
13
H50, H500). C NMR: d [ppm] (100.6 MHz, DMSO-d ): 177.24 (C O
[6] E. Brognara, I. Lampronti, G. Breveglieri, A. Accetta, R.
Corradini, A. Manicardi, M. Borgatti, A. Canella, C. Multineddu,
R. Marchelli, R. Gambari, Eur. J. Pharmacol. 2011, 672, 30–37.
–
–
6
–
quinolone); 166.15 (C O ester); 155.44 (phenoxy); 154.75 (C2);
–
142.66 (C6); 132.10 (C9); 129.45 (phenoxy); 126.68 (C7); 123.88
(phenoxy); 121.59 (C10); 119.05 (C5); 118.42 (phenoxy); 112.23
(C8); 106.61 (C3); 85.13 (C10); 80.35 (C40); 75.09 (C20); 68.92 (C30);
63.86 (C50). MALDI(þ)-MS (m/z) calcd.: 413.11 [C21H21N3O6], found:
413.43 [MþH]þ. HRMS (MþH): calcd.: 414.11834, found:
414.11901.
[7] I. Lampronti, N. Bianchi, C. Zuccato, A. Medici, P. Bergamini,
R. Gambari, Bioorg. Med. Chem. 2006, 14, 5204–5210.
[8] I. Lampronti, D. Martello, N. Bianchi, M. Borgatti,
E. Lambertini, R. Piva, S. Jabbar, M. S. Choudhuri,
M. T. Khan, R. Gambari, Phytomedicine 2003, 10, 300–308.
[9] F. Osti, F. G. Corradini, S. Hanau, M. Matteuzzi, R. Gambari,
Biological tests
Cell lines, culture conditions, and in vitro antiproliferative
activity assays
Haematologica 1997, 82, 395–401.
[10] N. Bianchi, C. Chiarabelli, M. Borgatti, C. Mischiati, E. Fibach,
R. Gambari, Br. J. Haematol. 2001, 113, 951–961.
Human CML K562 cells [8] were cultured in a humidified
atmosphere at 5% CO2 in RPMI‐1640 (Flow Laboratories, Irvine,
UK) supplemented with 10% fetal bovine serum: 100 units/mL
penicillin and 100 mg/mL streptomycin. The in vitro antiprolifer-
ative activities of quinolone nucleosides (9–12) were assayed as
follows. Cell number mL−1 was determined by using a model ZBI
Coulter Counter (Coulter Electronics, Hialeah, FL). Cells were
plated at an initial concentration of 3 × 104 cells/mL and the cell
number per milliliter was determined after 4 days, when
untreated cells are in the log phase of cell growth.
[11] N. Bianchi, F. Osti, C. Rutigliano, F. G. Corradini, E. Borsetti,
M. Tomassetti, C. Mischiati, G. Feriotto, R. Gambari, Br. J.
Haematol. 1999, 104, 258–265.
[12] N. Bianchi, F. Ongaro, C. Chiarabelli, L. Gualandi,
C. Mischiati, P. Bergamini, R. Gambari, Biochem. Pharmacol.
2000, 60, 31–40.
[13] C. Chiarabelli, N. Bianchi, M. Borgatti, E. Prus, E. Fibach,
R. Gambari, Haematologica 2003, 88, 826–827.
[14] S. Grant, Adv. Cancer Res. 1997, 72, 197–233.
[15] A. E. King, M. A. Ackley, C. E. Cass, J. D. Young, S. A. Baldwin,
Trends Pharmacol. Sci. 2006, 27, 416–425.
In vitro induction of erythroid differentiation
Erythroid differentiation of the four quinolone nucleosides (9–12)
was evaluated by counting benzidine positive cells after
suspending the cells in a solution containing 0.2% benzidine
in 0.5 M glacial acetic acid, 10% H2O2, as elsewhere described [10,
51]. Induction of differentiation was compared with that obtained
using well-established inducers of differentiation of K562 cells,
such as cytosine arabinose, mithramycin, angelicin, hydroxy-
urea, and butyric acid [52–55].
[16] M. Pastor-Anglada, F. J. Casado, in: Cancer Drug Discovery and
Development: Deoxynucleoside Analogs in Cancer Therapy (Ed.:
G. J. Peters), Humana Press Inc., New York 2007, pp. 1–28.
[17] M. Molina-Arcas, G. Moreno-Bueno, P. Cano-Soldado,
H. Hernandez-Vargas, F. J. Casado, J. Palacios, M. Pastor-
Anglada, Biochem. Pharmacol. 2006, 72, 1646–1656.
[18] W. Plunkett, P. Huang, C. E. Searcy, V. Gandhi, Semin. Oncol.
1996, 23, 3–15.
[19] A. Santoro, H. Bredenfeld, L. Devizzi, H. Tesch, V. Bonfante,
S. Viviani, F. Fiedler, H. S. Parra, C. Benoehr, M. Pacini,
G. Bonadonna, V. Diehl, J. Clin. Oncol. 2000, 18, 2615–2619.
Statistical analysis
All experiments were carried out in triplicate. Data were expressed
as means ꢀ SD. Differences were evaluated by one-way analysis of
variance (ANOVA) test completed by Dunnett’s test. Differences
were considered significant at ꢂꢂp < 0.01. The 50% inhibitory
concentration (IC50) was calculated by nonlinear regression
curve with the use of Prism Graphpad Prism version 4.0 for
Windows [GraphPad Software, San Diego, CA, USA (www.graph-
pad.com)].
[20] S. A. Johnson, Expert Opin. Pharmacother. 2001, 2, 929–943.
[21] D. Sampath, V. A. Rao, W. Plunkett, Oncogene 2003, 22, 9063–
9074.
[22] L. R. Peterson, Clin. Infect. Dis. 2001, 33, 180–186.
[23] G. Y. Lesher, E. J. Froelich, M. D. Gruett, J. H. Bailey,
R. P. Brundage, J. Med. Pharm. Chem. 1962, 91, 1063–1065.
[24] H. Koga, A. Itoh, S. Murayama, S. Suzue, T. Irikura, J. Med.
Chem. 1980, 23, 1358–1363.
We gratefully acknowledge Stefan Bernhardt for help with the HPLC
purification and Hanne Brill, Ilona Priess, and Andreas Münch for
mass spectra analysis.
[25] R. Wise, J. M. Andrews, L. J. Edwards, Antimicrob. Agents
Chemother. 1983, 23, 559–564.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–9
Synthesis and Antiproliferative Activity of Quinolone Nucleosides
9
[26] M. P. Wentland, D. M. Bailey, J. B. Cornett, R. A. Dobson,
[40] M. M. Adams, J. W. Bats, N. V. Nikolaus, M. Witvrouw,
Z. Debyser, J. W. Engels, Collect. Czech. Chem. Commun. 2006, 71,
978–990.
R. G. Powles, R. B. Wagner, J. Med. Chem. 1984, 27, 1103–1108.
[27] S. K. Bhanot, M. Singh, N. R. Chatterjee, Curr. Pharm. Des. 2001,
7, 311–335.
[41] N. V. Nikolaus, J. Božilović, J. W. Engels, Nucleosides Nucleotides
Nucleic Acids 2007, 26, 889–892.
[28] C. Sissi, M. Palumbo, Curr. Med. Chem. Anticancer Agents 2003, 3,
439–450.
[42] A. S. Guram, R. A. Rennels, S. L. Buchwald, Angew. Chem. Int.
Ed. 2005, 34, 1348–1350.
[29] E. Fibach, N. Bianchi, M. Borgatti, E. Prus, R. Gambari, Blood
2003, 102, 1276–1281.
[43] J. Louie, J. F. Hartwig, Tetrahedron Lett. 1995, 36, 3609–3612.
[44] S. Höger, Chem. Unserer Zeit 2001, 35, 102–109.
[30] A. D. Da Matta, A. M. R. Bernardino, G. A. Romeiro, M. R. P. De
Oliviera, M. C. B. V. De Souza, V. F. Ferreira, Nucleosides
Nucleotides 1996, 15, 889–898.
[45] M. Sato, T. Motomura, H. Aramaki, T. Matsuda,
M. Yamashita, Y. Ito, H. Kawakami, Y. Matsuzaki,
W. Watanabe, K. Yamataka, S. Ikeda, E. Kodama,
M. Matsuoka, H. Shinkai, J. Med. Chem. 2006, 49, 1506–1508.
[31] N. A. Al-Masoudi, Y. A. Al-Soud, M. Ehrmann, E. De Clercq,
Nucleosides Nucleotides 1998, 17, 2255–2266.
[32] J. P. Michael, C. B. de Koning, G. D. Hosken, T. V. Stanbury,
[46] R. G. Gould, W. A. Jacobs, J. Am. Chem. Soc. 1939, 61, 2890–
Tetrahedron 2001, 57, 9635–9648.
2895.
[33] M. Nasr, J. C. Drach, S. H. Smith, C. Shipman, Jr., J. H.
[47] M. Kidwai, P. Sapra, K. R. Bhushan, R. K. Saxena, R. Gupta,
Burckhalter, J. Med. Chem. 1988, 31, 1347–1351.
M. Singh, Monatsh. Chem. 2000, 131, 85–90.
[34] C. Périgaud, G. Gosselin, J. L. Imbach, Nucleosides Nucleotides
1992, 11, 903–945.
[48] U. Niedballa, H. Vorbrüggen, Angew. Chem. Int. Ed. 1970, 9,
461–462.
[35] A. De la Cruz, J. Elguero, P. Goya, A. Martinez, E. De Clercq, J.
Chem. Soc. Perkin Trans. 1, 1993, 845–849.
[49] U. Niedballa, H. Vorbrüggen, J. Org. Chem. 1974, 39, 3654–
3660.
[36] A. T. P. C. Gomes, A. C. Cunha, Md. R. M. Domingues, M. G.
P. M. S. Neves, A. C. Tom, A. M. S. Silva, Fd. C. Santos, M. C.
B. V. Souza, V. F. C. Ferreira, J. A. S. Cavaleiro, Tetrahedron
2011, 67, 7336–7342.
[50] E. Lager, P. Andersson, J. Nilsson, I. Pettersson, E. Østergaard
Nielsen, M. Nielsen, O. Sterner, T. Liljefors, J. Med. Chem. 2009,
49, 2526.
[51] R. Gambari, E. Fibach, Minerva Biotecnol. 2003, 15, 145–151.
[37] A. D. Da Matta, C. V. B. Dos Santos, H. D. Pereira, I. C.
D. P. Frugulhetti, M. R. P. de Oliveira, M. C. B. V. de Souza,
N. Moussatche, V. F. Ferreira, Heteroatom Chem. 1999, 10, 197–
202.
[52] R. Hoffman, M. J. Murnane, E. J. Benz, Jr., R. Prohaska,
V. Floyd, N. Dainiak, B. G. Forget, H. Furthmayr, Blood 1979,
54, 1182–1187.
[53] R. Cortesi, V. Gui, F. Osti, C. Nastruzzi, R. Gambari, Eur. J.
Haematol. 1998, 61, 295–301.
[38] S. Pasquini, C. Mugnaini, C. Tintori, M. Botta, A. Trejos,
R. K. Arvela, M. Larhed, M. Witvrouw, M. Michiels, F. Christ,
Z. Debyser, F. Corelli, J. Med. Chem. 2008, 51, 5125–5129.
[54] I. Lampronti, A. M. Saab, R. Gambari, Int. J. Oncol. 2006, 29,
989–995.
[39] C. V. B. D. Canuto, C. R. B. Gomes, I. P. Marques, L. V. Faro,
F. D. Santos, I. C. D. P. Frugulhetti, T. M. L. E. Souza,
A. C. Cunha, G. A. Romeiro, V. F. Ferreira, M. C. B. V. de Souza,
Lett. Drug Des. Discov. 2007, 4, 404–409.
[55] E. W. Iyamu, S. E. Adunyah, H. Fasold, K. Horiuchi, S. Baliga,
K. Ohene-Frempong, E. A. Turner, T. Asakura, Exp. Hematol.
2003, 31, 592–600.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com