Nucleolipids of Canonical Purine ?- D -Ribo-Nucleosides: Synthesis and Cytostatic/Cytotoxic Activities Toward Human and Rat Glioblastoma Cells

Article abstract of DOI:10.1002/open.201500197

We report on the synthesis of two series of canonical purine ?-d-ribonucleoside nucleolipids derived from inosine and adenosine, which have been characterized by elemental analyses, electrospray ionization mass spectrometry (ESI MS) as well as by ¹H and ¹³CNMR, and pH-dependent UV/Vis spectroscopy. A selection of the novel nucleolipids with different lipophilic moieties were first tested on their cytotoxic effect toward human macrophages. Compounds without a significant inhibitory effect on the viability of the macrophages were tested on their cytostatic/cytotoxic effect toward human astrocytoma/oligodendroglioma GOS-3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line. In order to additionally investigate the potential molecular mechanisms involved in the cytotoxic effects of the derivatives on GOS-3 or BT4Ca cells, we evaluated the induction of apoptosis and observed the particular activity of the nucleolipid ethyl 3-{4-hydroxymethyl-2-methyl-6-[6-oxo-1-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-1,6-dihydro-purin-9-yl]-tetrahydro-furo[3,4-d][1,3]dioxol-2-yl}propionate (8 c) toward both human and rat glioblastoma cell lines invitro. Nucleolipids combat cancer: We report the synthesis of two nucleolipid derivatives from inosine and adenosine with different lipophilic moieties. These have no cytotoxic effect on human macrophages based on invitro side-effect tests but have antiproliferative properties against malignant glioblastoma cell lines.

Full text of DOI:10.1002/open.201500197

DOI: 10.1002/open.201500197
Nucleolipids of Canonical Purine ß-d-Ribo-Nucleosides:
Synthesis and Cytostatic/Cytotoxic Activities Toward
Human and Rat Glioblastoma Cells
Christine Knies,[a] Katharina Hammerbacher,[b] Gabriel A. Bonaterra,*[b] Ralf Kinscherf†,[b] and
Helmut Rosemeyer†*[a]
ˇ
Dedicated to Prof. Dr. Jiri Zemlicka, Detroit, MI, USA.
We report on the synthesis of two series of canonical purine ß-
d-ribonucleoside nucleolipids derived from inosine and adeno-
sine, which have been characterized by elemental analyses,
electrospray ionization mass spectrometry (ESI MS) as well as
dendroglioma GOS-3 cells as well as against the rat malignant
neuroectodermal BT4Ca cell line. In order to additionally inves-
tigate the potential molecular mechanisms involved in the cy-
totoxic effects of the derivatives on GOS-3 or BT4Ca cells, we
evaluated the induction of apoptosis and observed the particu-
lar activity of the nucleolipid ethyl 3-{4-hydroxymethyl-2-
methyl-6-[6-oxo-1-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-1,6-di-
hydro-purin-9-yl]-tetrahydro-furo[3,4-d][1,3]dioxol-2-yl}propio-
nate (8c) toward both human and rat glioblastoma cell lines in
vitro.
1
by H and 13C NMR, and pH-dependent UV/Vis spectroscopy. A
selection of the novel nucleolipids with different lipophilic moi-
eties were first tested on their cytotoxic effect toward human
macrophages. Compounds without a significant inhibitory
effect on the viability of the macrophages were tested on their
cytostatic/cytotoxic effect toward human astrocytoma/oligo-
Introduction
In a series of precedent publications we and others have dem-
onstrated that the cancerostatic/cancerotoxic activity of pyrimi-
dine ß-d-ribonucleoside antimetabolites such as 5-fluorouri-
dine and 6-azauridine towards different human tumor cell
lines[1,2] as well as neurobiological[3] and antiviral activities[4,5]
can be significantly improved by lipophilization. Also, regular,
canonical pyrimidine ß-d-ribonucleosides such as uridine
and 5-methyluridine acquire a surprisingly high antitumor in
vitro activity upon covalent hydrophobization.[6,7] The posi-
tioning and type of the lipophilic residues are hereby of deci-
sive importance. It has been shown that, in particular, the
introduction of an ethyl levulinate group at the O-2’,3’-hy-
droxyls in form of a cyclic ketal and, additionally, a farnesyl ses-
quiterpene moiety at N(3) leads to compounds with significant
activity.[2]
In this manuscript, we extend our study to purine ß-d-ribo-
nucleoside nucleolipids, particularly to inosine and adenosine
derivatives. Again, a selection of the compounds was tested
with respect to the viability/survival of phorbol 12-myristate
13-acetate (PMA)-differentiated human THP-1 macrophages
when treated with these compounds.[2] Those which proved to
be nontoxic for the immune cells were then further tested on
their cytostatic/cytotoxic in-vitro activity towards human astro-
cytoma/oligodendroglioma GOS-3 cells, as well as against rat
malignant neuroectodermal BT4Ca cells.
[a] C. Knies,+ Prof. Dr. H. Rosemeyer
Organic Chemistry I–Bioorganic Chemistry
Institute of Chemistry of New Materials
University of Osnabrück, Barbarastr. 7, 49069 Osnabrück (Germany)
Results and Discussion
Synthesis
E-mail: Helmut.Rosemeyer@uni-osnabrueck.de
[b] K. Hammerbacher,+ Dr. G. A. Bonaterra, Prof. Dr. R. Kinscherf
Anatomy and Cell Biology, Department of Medical Cell Biology
University of Marburg, Robert-Koch-Straße 8, 35032 Marburg (Germany)
E-mail: gabriel.bonaterra@staff.uni-marburg.de
Starting from adenosine (1), its ethyl levulinate derivative 3
was prepared according to
a
well-known procedure
(Scheme 1).[8] However, in contrast to older publications, we
found that this ketal formation resulted in the formation of
a diastereoisomeric mixture (1R)/(1S) with a ratio of about 10:1
in all cases.
[+] These authors contributed equally to this publication.
[†] Senior authors
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/open.201500197.
Ketal formation of adenosine with nonadecan-10-one gave
the nucleolipid 5. Both O-2’,3’ ketals (3 and 5) were then sub-
mitted to an enzymatic deamination using adenosine deami-
nase (from calf intestine). It could be clearly shown that com-
pound 3 could be deaminated within 72 h yielding the inosine
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This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited and is not used for commercial purposes.
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Scheme 1. Stepwise lipophilization of adenosine (1) and inosine (2) at their O-2’,3’-position and at the nucleobase. Reagents and conditions: a) adenosine
deaminase, H2O, rt, 24 h, 100%; b) H3CÀ(CH2)8ÀC(=O)À(CH2)8ÀCH3, (EtO)3CH, 4m HCl in 1,4-dioxane, DMF, rt, 24 h, 5: 39% , 6: 35%; c) H3CÀC(=O)À(CH2)2À
C(=O)OC2H5, (EtO)3CH, 4m HCl in 1,4-dioxane, DMF, rt, 24 h, 71%; d) farnesyl bromide (for 7), D2-isopentenyl bromide (for 8a), geranyl bromide (for 8b), or
farnesyl bromide (for 8c), K2CO3, DMF, rt, 24 h, 7:61%, 8a: 48%, 8b: 40%, 8c: 57% ; e) farnesyl bromide, K2CO3, DMF, 308C, 30 min, then rt, 24 h, 37%.
derivative 4,[9] while 5 could not be deaminated to 6. The
latter was obtained by direct ketal formation of inosine (2)
with nonadecan-10-one.
inspection of the NMR spectra, particularly of the 13C NMR
spectra, revealed that compound 3 was formed as a diastereo-
isomeric (1R)/(1S)-mixture (for an example of the diastereoiso-
mer structures, see Scheme 2), while the subsequent deamina-
tion product 4 proved to be the diastereoisomerically almost
pure (1R) derivative. This might be traced back to the following
reasons: 1) It has been shown earlier that the enzymatic deam-
ination of adenosine which has been ketalized at the 2’,3’-O
position with unsymmetrical ketones such as pentan-2-one[10]
to the corresponding (1R) and (1S) 2’,3’-O-(1-methylbutylide-
All novel compounds were characterized by elemental analy-
ses, high-resolution electrospray ionization mass spectrometry
1
(HR ESI MS) as well as by H and 13C NMR, and pH-dependent
UV/Vis spectroscopy. Assignment of 13C NMR resonances was
made with the help of DEPT-135 as well as by gradient-select-
ed homo- and heteronuclear correlation spectroscopy (Bruker
pulse programs, 1H,13C-HSQCETGP; 1H,1H-COSYGPSW). Careful
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Scheme 2. Dimroth rearrangement of compounds 13a–c, prepared from
compound 3, yielding the N(6)-alkylated compounds 14a–c. Reagents and
conditions: a) D2-isopentenyl bromide (for 13a), geranyl bromide (for 13b),
or farnesyl bromide (for 13 c), DMF, BaCO3, Ar atmosphere, rt, 24 h, 13a:
63%, 13b: 58%, and 13c: 67%; b) (CH3)2NH in H2O (1m), rt, 20 h, 14a: 62%,
14b: 23%, and 14c: 38%.
Scheme 3. Ketal formation of inosine (2) with 4-oxopentyl 4-methylbenzoate
yielding almost equimolar amounts of nonseparated (1R)-9 (55%) and (1S)-9
(45%) diastereoisomers.[12] Reagents and conditions: 4-oxopentyl 4-methyl-
benzoate, (EtO)3CH, 4m HCl in 1,4-dioxane, DMF, rt, 24 h.
ne)adenosines occurs with significantly different Michaelis–
Menten kinetics; the (1S)-configured ketal is deaminated at an
8.5-fold lower vmax rate than the (1R)-configured isomer.[10]
2) The deamination product 4 was isolated by crystallization
which might lead to a preferred precipitation of the corre-
sponding (1R) product. In contrast to this, ketal formation of
inosine (2) with ethyl levulinate in the presence of triethylor-
thoformate[9,11] leads to a diastereoisomeric mixture of com-
pound 4 [(1R):(1S)ꢀ10:1] (Schemes 1 and 3).
In the following, we lipophilized the inosine nucleolipid 6
further at N(1) by farnesylation (dimethylformamide [DMF],
K2CO3)[15] and obtained compound 7. It could be shown that
the alkylation occurs without 5’-OH protection which might be
substantially advantageous for an antitumor activity as the
latter requires probably an intracellular phosphorylation of the
5’-OH group. Because it had been recently shown that a lipo-
philization of pyrimidine ß-d-ribonucleosides at the O-2’,3’ po-
sition as well as at N(3) of the pyrimidine base leads to nucleo-
lipids with a pronounced and selective cytostatic/cytotoxic in
vitro activity toward various human tumor cell lines,[2] we now
converted also the inosine derivative 4 [pure (1R)-diastereoiso-
mer, prepared by enzymatic deamination from compound 3]
to the corresponding N(1)-prenylated inosine nucleolipids 8a–
c. In the case of the preparation of compounds 8a,b by-prod-
ucts, such as probably formed O-alkylated compounds, were
chromatographically removed and not further characterized.
Only in the case of the farnesylation of compound 4, the reac-
tion was studied in more detail; it was performed at two differ-
ent temperatures: 1) At room temperature (208C), the alkyla-
tion afforded the N(1)-prenylated derivatives 8c (kinetic reac-
tion control, see Experimental Section). 2) Alkylation at already
slightly elevated temperatures (40–558C), however, gave, after
work-up, the N(1)- as well as further O(4)-prenylated deriva-
tives such as 8d (thermodynamic reaction control) and another
Interestingly, the 13C NMR spectrum of compound 4, pre-
pared by the latter method, exhibits characteristically increas-
ing Dd values [(1R)–(1S)] for the C(1’), C(4’), C(2’), and C(3’) car-
bons and a strong decrease again for C(5’) (Figure 1). The
graph shown in Figure 1 demonstrates almost identical chemi-
cal shift differences of compound 4 as well as of the analogous
compound 9[12]—both adopting an anti-conformation at the N-
glycosylic bond. The formation of an almost equimolar mixture
of (1R) and (1S) diastereoisomers of a ketal formation reaction
of inosine with 4-oxopentyl 4-methylbenzoate has been found
already recently.[12] It was corroborated also for analogous reac-
tions with other nucleosides which will be the subject of
a forthcoming publication. The almost identical chemical shift
differences, which have already been observed earlier for pyri-
midine ß-d-ribonucleoside ketals[7] might be the result of the
interworking of various CÀO and CÀC magnetic anisotropy ef-
fects[13] of ketal moiety bonds (Figure S1 in the Supporting In-
formation), for example of {C(acetal)ÀO} and {CH2(acetal)À one, the structure of which proved to be more complicated.
CH2(C=O)} bonds, within (1R)-O-2’,3’-[1-(2-carboxyethyl)ethyli-
den]adenosine.[14].Others such as {C(acetal)ÀCH2(acetal)} aniso-
tropy effects may be counterproductive.
The general structure of the O-alkylated derivatives was corro-
borated by pH-dependent UV/Vis spectroscopy. Tentatively, we
postulate that the O(6)-farnesylated nucleolipid 8d reacts with
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Figure 1. 13C NMR chemical shift differences of the glyconic moiety resonances of compounds 4 and 9, both as an unseparated diastereoisomeric mixture (A),
and the corresponding (1R)-isomers are displayed (B and C).
an excess of farnesyl bromide (10) via the intermediate 11 to
the O(6)-triterpenyl derivative 12 (Scheme 4)—an inosine nu-
cleolipid carrying a squalene-analogous N(1)-side chain, which
has been formed by a head–tail addition of two farnesyl resi-
dues. This is underlined by pH-dependent UV/Vis and NMR
spectroscopy (1H, 13C NMR) as well as by HR ESI MS spectrome-
try.
Next, we alkylated the adenosine nucleolipid 3[8] with three
different prenyl bromides and obtained the corresponding
N(1)-alkylated salts 13a–c (Scheme 2). These were submitted
to Dimroth rearrangements[16,17] with an aqueous dimethyla-
mine solution[18] which yielded the adenosine nucleolipids
14a–c. By this way, we were able to synthesize chain-extended
analogues of the nucleoside antimetabolite N(6)-isopentenyla-
denosine {=N6-(D2-isopentenyl)adenosine}[19] carrying addition-
ally an O-2’,3’-ethyllevulinate ketal group without saponifica-
tion of the ester. In an earlier publication we have reported the
synthesis of an N(6)-isopentenyladenosine derivative with an
O-2’,3’-levulinate moiety using 1 n aq NaOH.[20] All compounds
were characterized by HR ESI MS, elemental analyses, as well
1
as by H and 13C NMR and UV/Vis spectroscopy.
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Scheme 4. Side reactions occuring upon farnesylation of inosin-O-2’,3’-ketal 4. Reagents and conditions: a) farnesyl bromide, K2CO3, DMF, rt, 24 h, 57%; b) far-
nesyl bromide, K2CO3, DMF, 308C, 30 min, then r.t., 24 h; c) excess of farnesyl bromide, 24 h, 558C, 12: 37%. REL: ribosylethyllevulinate.
Lipophilic properties of purine ß-d-ribonucleosides and
their nucleolipids
Table 1. Translation of plain compound numbers into the NS/NL-nomen-
clature.[23]
Applying the ALOGPS v.3.01 program,[21,22] the 10LogPow values
of the nucleolipids, as well as of their precursor molecules,
were calculated; for this purpose we used the eadmet.com/de/
physprop.php site with ePhysChem that contains the program
mentioned. The results are shown in Figure 2A,B. The figures
clearly demonstrate the possibility of a fine tuning of the com-
pounds’ lipophilicity by the introduction of stepwise elongated
prenyl side chains to both adenosine and inosine.
In addition to 10LogPow calculations, we have also measured
such data experimentally for those four compounds which
have been tested biologically (see Experimental Section). A
comparison of calculated and experimental data can be seen
in Table 1.
Compound num-
bers
NS/NL code Compound num-
NS/NL
code[a]
[a]
bers
1
2
3
4
5
6
7
8a
8b
8c
8d
NS_5.0.0.0
NS_6.0.0.0
NL_5.1.0.0
NL_6.1.0.0
NL_5.3.0.0
NL_6.3.0.0
NL_6.3.13.0
NL_6.1.11.0
NL_6.1.12.0
NL_6.1.13.0
NL_6.1.63.0
9
10
11
12
13a
13b
13c
14a
14b
14c
NL_6.6.0.0
N.a.n.
N.a.n.
N.a.n.
NL_5.1.11.0
NL_5.1.12.0
NL_5.1.13.0
NL_5.1.61.0
NL_5.1.62.0
NL_5.1.63.0
[a] According to Ref. [23]. N.a.n.: not a number.
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Figure 3. Viability/survival of differentiated human THP-1 macrophages after
48 h of incubation with 5-FU (5-fluorouridine), or its derivatives 5, 6, or 8c.
Values are given [in % survival of control (incubation with medium
alone=100% survival] as meanÆSEM; ***p<0.001 vs. negative control;
n=4.
and 91.2% (p<0.001), in comparison with the control
(Figure 4).
Moreover, incubation (48 h) of the rat malignant neuroecto-
dermal BT4Ca cells with 5-fluorouridine (1.56, 3.12, 6.25, 12.5,
25, or 50 mm) resulted in significant (p<0.001), 60.3, 58.7, 65.1,
59.5, 58.8, and 68.1% inhibitions of the viability/survival when
compared with the control. At concentrations of 25 mm and
50 mm, the derivative 8c significantly (p<0.001) inhibited the
viability/survival by 58.6% and 81.8%, and at a concentration
of 12.5 mm, viability/survival was (not significantly) inhibited by
11.9%, in comparison with the control (Figure 5).
Figure 2. 10LogPow values of A) purine ß-d-ribonucleosides adenosine (5) and
B) inosine (6), as well as their nucleolipids.
Biological results
Using an in vitro model to differentiate between anticancer
properties and side effects, we tested the cytotoxic effects on
the viability of PMA-differentiated human THP-1 macrophages
after treatment for 48 h. At concentrations of 6.25, 12.5, 25, or
50 mm 5-fluorouridine, there was a significant inhibition of the
viability/survival by 11.9% (p<0.001), 8.4% (p<0.05), 13.7%
(p<0.001), 12.14% (p<0.05) compared with negative control
(Figure 3). The purine derivative 8c did not exhibit any signifi-
cant inhibitory effect on viability/survival of THP-1 macrophages
(Figure 3). At a concentration of 50 mm, the derivatives 5 and
6[23] revealed significantly (p<0.001) cytotoxic effects of 93.3%
and 86.5%; additionally, the derivative 5 significantly (p<
0.001) showed cytotoxic effects of 69.9% at 25 mm, in compari-
son with the control (Figure 3). Because the derivative 8c re-
vealed no or only marginal cytotoxic effects on differentiated
human THP-1 macrophages, the effects of this substance—in
comparison with the positive control (5-fluorouridine)—was
tested in human astrocytoma/oligodendroglioma GOS-3 and
rat malignant neuroectodermal BT4Ca cells. Incubation (48 h)
of the human astrocytoma/oligodendroglioma GOS-3 cells
with 5-fluorouridine, at concentrations of 1.56, 3.12, 6.25, 12.5,
25, or 50 mm, resulted in significant (p<0.001) 45.5, 54.4, 51.6,
54.0, 53.7, and 59.0% inhibitions of the viability/survival,
whereas incubation with derivative 8c at 25 mm and 50 mm sig-
nificantly inhibited the viability/survival by 28.1% (p<0.01)
In order to investigate additionally the potential molecular
mechanisms involved in the cytotoxic effects of the derivatives
on GOS-3 or BT4Ca cells, induction of apoptosis was evaluated.
At a concentration of 50 mm, the derivative 5-fluorouridine sig-
Figure 4. Viability/survival of human astrocytoma/oligodendroglioma GOS-3
cells after 48 h of incubation with 5-FU (5-fluorouridine, positive control) or
its derivative 8c. Values are given [in % survival of control (incubation with
medium alone=100% survival] as meanÆSEM; **p<0.01, ***p<0.001 vs.
negative control (medium alone); n=5.
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Figure 5. Viability/survival of rat malignant neuroectodermal BT4Ca cells
after 48 h of incubation with 5-FU (5-fluorouridine), or its derivative 8c. [in
% survival of control (incubation with medium alone=100% survival] as
meanÆSEM; ***p<0.001 vs. negative control; n=5.
nificantly (p<0.01) enhanced the percentage of apoptotic
GOS-3 cells by 8.6%, when compared with the control (Fig-
ure 6A,C). Moreover, a significant (p<0.05) 63.6% and 62.0%
decrease of the cell number was found using 25 mm and 50 mm
of the derivative 5-fluorouridine (Figure 6B,C ). At a concentra-
tion of 50 mm, the derivative 8c significantly (p<0.001) in-
duced apoptosis by 94.9% (Figure 6A,C); additionally, a signifi-
cant (p<0.05 and p<0.001) 45.7% and 82.4% decrease of the
cell number was observed by 25 mm and 50 mm treatment, in
comparison with the control (Figure 6B,C).
Treatment of BT4Ca cells with 25 mm or 50 mm of 5-fluorouri-
dine induced an increase of apoptosis by 5.2% (p<0.01) and
14.7% (p<0.001) (Figure 7A,C), and decreased the cell number
by 93.6% (p<0.001) and 95.7% (p<0.001) in comparison with
the control (Figure 7B,C). Treatment of BT4Ca cells with the de-
rivative 8c (50 mm) significantly (p<0.001) enhanced the per-
centage of apoptosis by 95.7%, in comparison with the control
(Figure 7A,C). Moreover, treatment of BT4Ca cells with the de-
rivative 8c at 25 mm or 50 mm significantly (p<0.001) de-
creased the cell number by 56.5% and 97.3%, in comparison
with the control (~100% cells) (Figure 7B,C).
Additional investigations of the protein expression (western
blot) of the ubiquitin-binding autophagic adaptor p62/SQSMT1
(hereafter p62) showed a significant (p<0.01) activation being
260% higher than the negative control (100%) after treatment
with 25 mm 8c (Figure 8A,C). Proliferating cell nuclear antigen
(PCNA) expression showed a significant (p<0.05) inhibition by
84.4% or 73.1%, which was, however, not significant after
treatment with 12.5 mm or 25 mm of 5-fluorouridine, respective-
ly (Figure 8A,C). Whereas treatment with 12.5 mm and 25 mm
8c significantly (p<0.01) inhibited the PCNA expression in
GOS-3 cells by 76.6% and 96.8% (Figure 8B,C). Furthermore,
treatment of BT4Ca cells with 12.5 mm 5-fluorouridine showed
a significant (p<0.01) 67.7% inhibition, whereas the effect of
25 mm was nonsignificant when compared with the control.
Treatment with 12.5 mm of 8c showed a significant (p<0.01)
Figure 6. Treatment of human GOS-3 cells with 5-FU (5-fluorouridine), or its
derivative 8c after 48 h of incubation. Effects of 5-FU or its derivative 8c on
A) apoptosis rate, B) total cell number, C) morphological/quantitative
changes of the apoptosis in GOS-3 cells observed by Hoechst 33342 (total
cell count) or YOPRO-1 (apoptosis) staining, using a fluorescence microscope
and phase contrast. Values in %apoptosis (A), or in % of control (100% of
cell number) with medium alone (B), are given as meanÆSEM; *p<0.05,
**p<0.01, ***p<0.001 vs. negative control; n=4. Scale bar: 50 mm; magnifi-
cation”100.
50.2% inhibition, but 25 mm increased significantly (p<0.05)
49.5% the protein expression of p62/SQSMT1 when compared
with the control (Figure 9A,C). Furthermore, after treatment of
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Figure 8. Effects of 48 h treatment of GOS-3 cells with 5-FU (5-fluorouridine),
or its derivative 8c shown as a western blot of A) autophagy (SQSTM1/p62)
and B) proliferation (PCNA) markers quantified by densitometric analysis.
Values expressed in % of negative control (100%) are given as meanÆSEM;
*p<0.05 and **p<0.01 vs. control; n=3. C) Representative western blots of
p62/SQSTM1 and PCNA are shown.
Figure 7. Treatment of rat BT4Ca cells with 5-FU (5-fluorouridine), or its de-
rivative 8c after 48 h of incubation. Effects of 5-FU or its derivative 8c on
A) apoptosis rate, B) total cell number, C) morphological/quantitative
changes of the apoptosis in BT4Ca cells observed by Hoechst 33342 (total
cell count) or YOPRO-1 (apoptosis) staining, using a fluorescence microscope
and phase contrast. Values in %apoptosis are given as meanÆSEM.
***p<0.001 vs. negative control (A); or in % of control (100%) with medium
alone (B); n=4. Scale bar: 50 mm; magnification”100.
or 25 mm of the derivative 8c, we found a significant (p<0.01)
12.6% or 80.4.3% inhibition of the PCNA protein expression
when compared with the control (Figure 9B,C).
Experimental Section
Nomenclature
For the general numbering of nucleosides and nucleolipids,
a novel denomination system was developed which allows an easy
comparison of compound data among the various publications of
our groups and which is disclosed in ref. [23]. Moreover, it was de-
posited sustainably in the repository of the library of the University
BT4Ca cells with 12.5 mm or 25 mm 5-fluorouridine, we found
an (insignificant) 54.9% and 42.1% inhibition of PCNA expres-
sion on protein level (Figure 9B,C). When treated with 12.5 mm
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of the NS/NL nomenclature to the plain compound numbers used
throughout the text, schemes, figures see Table 1.
Chemistry
General Remarks: All chemicals were purchased from Sigma–
Aldrich (Deisenhofen, Germany) or TCI-Europe (Zwijndrecht, Bel-
gium). Solvents were of laboratory grade and were distilled before
use. Column chromatography was performed on silica gel 60. Thin-
layer chromatrography (TLC) was performed using aluminum
sheets and silica gel 60 F254; 0.2 mm layer (Merck, Germany). NMR
spectra (1H, 13C, DEPT-135) were obtained using an AMX-500 instru-
ment (Bruker, Rheinstetten, Germany); 1H: 500.14 MHz, 13C:
125.76 MHz; chemical shifts (d) are reported in ppm referenced to
an internal standard of residual proteosolvent [D6]DMSO (2.50,
39.50 ppm, rel. to tetramethylsilane (TMS) as internal standard).
Multiplicity is quoted as br (broad), s (singlet), d (doublet), t (trip-
let), q (quartet), quint (quintet), m (multiplet), and Yt (pseudotrip-
let), dd (doublet of doublet), ddd (doublet of doublet of doublet).
J values are reported in Hz. 2D [1H,1H] and [1H,13C] correlation spec-
tra (heteronuclear single quantum coherence, HSQC) and Cosy-
Long-Range spectra (pulse program: cosygpmfph) were measured
with the same instrument.
Sample preparation was performed as follows: an appropriate
amount of compound (usually 20–25 mg) was dissolved in
[D]6DMSO (0.5 mL) and placed in the NMR quartz tube (diameter,
5 mm). Before measurement the solutions were degassed by ultra-
sonication for several minutes. Number of scans: 1H: 64, 13C:
12.000, DEPT-135: 5.000. ESI MS was performed using a Bruker Dal-
tronics Esquire HCT instrument (Bruker Daltronics, Leipzig, Germa-
ny); ionization was performed with a 2% aq. formic acid (HCOOH)
solution. UV/VIs spectra were obtained using a Cary 1E spectropho-
tometer (Varian, Darmstadt, Germany). Compound samples of
about 1 mg were dissolved either in MeOH or an appropriate
buffer solution (pH 3, 7, or 9, 100 mL each). Aliquots of 1 mL of the
fully dissolved compounds (warming, ultrasonication) were subject-
ed to UV/Vis spectrometry in MT4 quartz cuvettes (Hellma, Darm-
stadt, Germany). Elemental analyses (C, H, N) were performed on
a
VarioMICRO instrument (Fa. Elementar, Hanau, Germany).
10LogPOW values were calculated using the http://eadmet.com/de/
physprop.php website with ePhysChem that contains ALOGPS v.3.0.
Experimental determination of 10LogPow values of compounds were
performed as follows: samples of compounds 5-fluorouridine (NS_
4.0.0.0), 6 (NL_5.3.0.0), 8c (NL_6.1.13.0), 6 (NL_6.3.0.0) (2 mg, each)
were dissolved in a heterogenic mixture of n-butanol (25 mL) and
H2O (25 mL) by ultrasonication (10 min) under slightly warming.
After separation of the layers from each phase aliquots of 1 mL
were withdrawn, and their UV spectra were run in 1 cm quartz cuv-
Figure 9. Effects of 48 h treatment of BT4Ca cells with 5-FU (5-fluorouridine),
or its derivative 8c. Western blot of (A) autophagy [p62/SQSTM1] and
(B) proliferation [PCNA] markers quantified by densitometric analysis. Values
expressed in% of negative control (=100%) are given as mean+SEM.
*p<0.05 and **p<0.01 (by T-TEST) significance vs. control. Representative
western blots of p62/SQSTM1 and PCNA (C) are shown; n=3 independent
experiments.
ettes. From the ratio of maximal extinctions of both layers at lmax
,
the corresponding 10LogPow values were calculated and compared
of Osnabrück under the following unique registration number
(URN) and URL:
with increment-based calculations (Table 2).
https://repositorium.uni-osnabrueck.de/handle/urn:nbn:
de:gbv:700-2015110413639.
General synthetic methods
The complete characterization data for all synthesized compounds
mentioned below can be found in the Supporting Information.
Nucleolipids are abbreviated by NL, nucleosides by NS. The first
number refers to the nucleoside. The second number refers to the
moiety at the 2’,3’-position at the glyconic ring; cyclic moieties are
abbreviated by “cycl” before the number. The third number refers
to the lipophilic moiety at the base [N(3) for pyrimidines, N(1) for
purines]. The fourth number refers to a lipophilic moiety at the 5’-
O-position. Identical residues carry the same number; “0” stands
for a molecule without a residue at this position. For a translation
Ethyl 3-[4-(6-amino-purin-9-yl)-6-hydroxymethyl-2-methyl-tetra-
hydro-furo[3,4-d][1,3]dioxol-2-yl]-propionate (3, NL_5.1.0.0,[8]
diastereoisomeric mixture): The compound was prepared follow-
ing a previously described procedure,[8] but with slight modifica-
tions and supported by further analytical data. To a solution of an-
hydrous adenosine (1, NS_5.0.0.0,[23] 1.38 g, 5 mmol) in dry and
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87:13). Evaporation of the main zone gave the title compound as
a colorless oil (776 mg, 1.46 mmol, 39%).
Table 2. Comparison of calculated and UV/Vis-measured 10LogPow values
of 5-fluorouridine and compounds 5, 6, and 8c used for in vitro tests.
9-(6-Hydroxymethyl-2,2-dinonyl-tetrahydro-furo[3,4-d][1,3-
dioxol-4-yl)-1,9-dihydro-purin-6-one (6, NL_6.3.0.0): To a solution
of anhydrous inosine (2, 1.0 g, 3.72 mmol) in dry DMF (15 mL), non-
adecan-10-one (2.1 g, 7.44 mmol), triethylorthoformate (1.0 g,
5.58 mmol), and 4m HCl in 1,4-dioxane (3.4 mL) were added. The
reaction mixture was stirred for 48 h at ambient temperature
under exclusion of moisture. Then, the mixture was partitioned be-
tween CH2Cl2 (350 mL) and a saturated aqueous NaHCO3 solution
(50 mL). The organic layer was washed with H2O (3”100 mL), and
the combined aqueous phases were re-extracted with CH2Cl2 (2”
25 mL). The combined organic layers were dried (Na2SO4), filtered,
and evaporated. The residue was dried o/n in high vacuum and
then submitted to column chromatography (SiO2 60H, column: 6”
14 cm, CH2Cl2/MeOH 9:1). Evaporation of the main zone afforded
the title compound as a colorless oil (700 mg, 1.314 mmol, 35%).
Compound
10LogPow measured
10LogPow calculated[21]
5-Fluorouridine
À0.61
ꢁ4.00
ꢁ3.50
ꢁ3.74
À1.3Æ0.38
5.1Æ0.74
5.3Æ0.74
3.5Æ0.74
5
6
8c
Values are for single measurements.
amine-free dimethylformamide (DMF, 10 mL), ethyl levulinate
(1.42 mL, 10 mmol), triethylorthoformate (1.65 mL, 10 mmol) and
4m HCl in 1,4-dioxane (2 mL) were added. After stirring for 27 h at
ambient temperature, the reaction mixture was partitioned be-
tween CH2Cl2 (75 mL) and a saturated aqueous NaHCO3 solution
(30 mL). The aqueous phase was washed with CH2Cl2 (2”25 mL),
and the combined organic layers were evaporated on a rotary
evaporator. The resulting oil was co-evaporated repeatedly with
CH2Cl2 in order to remove residual DMF. The product was precipi-
tated by addition of dry diethyl ether (Et2O), filtered, and dried in
high vacuum overnight yielding the title compound as colorless
crystals (1.24 g, 3.15 mmol, 71%; diastereoisomeric mixture, [1R]/
[1S]=12:1).
9-(6-Hydroxymethyl-2,2-dinonyl-tetrahydro-furo[3,4-d]
[1,3]dioxol-4-yl)-1-(3,7,1)-trimethyl-dodeca-2,6,10-trienyl)-1,9-di-
hydro-purin-6-one (7, NL_6.3.3.0): To a solution of compound 6
(593 mg, 1.113 mmol) in dry DMF (6 mL), K2CO3, (390 mg,
2.88 mmol) was added, and the suspension was stirred for 30 min
at ambient temperature. Thereupon, farnesyl bromide (0.35 mL,
1.17 mmol) was added dropwise under an N2 atmosphere while
stirring. After 24 h, K2CO3 was filtered off and washed repeatedly
with CH2Cl2 (2”25 mL). The filtrate and washings were evaporated
on a rotary evaporator; the resulting oil was dried in high vacuum
for 24 h. The residue was submitted to column chromatography
(SiO2 60H, column: 6”12 cm, CH2Cl2/MeOH 97:3). Evaporation of
the main zone afforded the title compound as a colorless oil
(499.2 mg, 0.677 mmol, 61%).
Ethyl 3-[4-hydroxymethyl-2-methyl-6-(6-oxo-1,6-dihydro-purin-
9-yl)-tetrahydro-furo[3,4-d][1,3]dioxol-2-yl]-propionate (4, NL_
6.1.0.0[9,11,23]): A) By enzymatic deamination of 3: Compound 3
(0.94 g, 2.39 mmol) was dissolved in H2O (27 mL), and adenosine
deaminase (90 units, calf intestine)—dissolved in 0.9 mL of glycer-
ol—was added. The reaction mixture was stirred at ambient tem-
perature for 72 h (TLC monitoring). The resulting solution was
evaporated on a rotary evaporator, leaving colorless crystals of the
title compound as pure (1R)-4 diastereoisomer (0.94 g, 2.39 mmol,
100%). B) By Direct Ketal Formation of Inosine: Anhydrous inosine
(2, 1.0 g; 3.73 mmol) was dissolved in dry DMF (18 mL). Then, ethyl
levulinate (1.05 mL; 7.41 mmol), triethylorthoformate (0.92 mL;
5.60 mmol), and 4m HCl in 1,4-dioxane (3.5 mL) were added, and
the reaction mixture was stirred for 26 h at rt. The reaction mixture
was partitioned between CH2Cl2 (175 mL) and a saturated conc. aq.
NaHCO3-solution (5 mL). The aqueous phase was washed twice
with CH2Cl2 (80 mL, each). The combined organic layers were
evaporated and repeatedly co-evaporated from CH2Cl2 to remove
residual DMF. The oily residue was dried in high vacuum o/n at
408C and subsequently purified by column chromatography (SiO2
60H, column: 6.5”14 cm; CH2Cl2/MeOH; 85:15; v/v). From the
main zone, the title compound was isolated as a white powder
(diastereoisomeric mixture, 1.108 g, 2.81 mmol, 75%). The material
proved to be identical with an authentic sample in all respects,
except for the NMR data.[9,11]
Ethyl 3-{4-hydroxymethyl-2-methyl-6-[1-(3-methyl-but-2-enyl)-6-
oxo-1,6-dihydro-purin-9-yl]-tetrahydro-furo[3,4-d][1,3]dioxol-2-
yl}-propionate (8a, NL_6.1.11.0,[23]): To a solution of anhydrous
(dried at 808C overnight), stereochemically pure compound (1R)-4
(0.35 g, 0.89 mmol) in dry DMF (10 mL), dry K2CO3 (0.49 g,
3.55 mmol) was added. The suspension was stirred for 30 min at
ambient temperature. Subsequently, isopentenyl bromide
(0.95 mmol) was added dropwise under Ar atmosphere and under
exclusion of light and moisture. The reaction mixture was stirred
for 60 h at rt. The salt was then filtered off and washed with CH2Cl2
(2”25 mL); the filtrate and washings were combined and parti-
tioned between H2O (30 mL) and CH2Cl2 (40 mL) in a separation
funnel. The organic phase was pooled, and the aqueous was
washed with CH2Cl2 (2”25 mL). The combined organic layers were
dried (Na2SO4), filtered, and evaporated on a rotary evaporator. Re-
sidual DMF was removed by repeated co-evaporation with CH2Cl2
and subsequent drying in high vacuum. Column chromatography
(SiO2 60H, column: 5”6.5 cm, CH2Cl2/MeOH 98:2) gave the title
compound as a colorless oil (200 mg, 0.43 mmol, 48%)
6-(6-Amino-purin-9-yl)-2,2-dinonyl-tetrahydro-furo[3,4-d]
[1,3]dioxol-4-yl]-methanol (5, NL_5.3.0.0[23]): To a solution of an-
hydrous adenosine (1.0 g, 3.74 mmol) in dry DMF (15 mL) nonade-
can-10-one (2.1 g, 7.48 mmol), triethylorthoformate (1.0 g,
5.61 mmol), and 4m HCl in 1,4-dioxane (3.4 mL) were added. The
reaction mixture was stirred for 48 h at rt and then partitioned be-
tween CH2Cl2 (175 mL) and a sat. aq. NaHCO3 solution (50 mL). The
organic layer was washed with H2O (3”100 mL), and the combined
aqueous layers were re-extracted with CH2Cl2 (25 mL). The com-
bined organic phases were evaporated, and the oily residue was
dried in high vacuum overnight and then submitted to column
chromatography (SiO2 60H, column: 6”12 cm, CH2Cl2/MeOH
Ethyl
3-{4-[1-(3,7-dimethyl-octa-2,6-dienyl)-6-oxo-1,6-dihydro-
2-methyl-tetrahydro-furo[3,4-d]
purin-9-yl]-6-hydroxymethyl-
[1,3]dioxol-2-yl}-propionate (8b, NL_6.1.12.0,[23]): To a solution of
anhydrous (dried at 808C overnight), stereochemically pure com-
pound (1R)-4 (0.35 g, 0.89 mmol) in dry DMF (10 mL), dry K2CO3
(0.49 g, 3.55 mmol) was added. The suspension was stirred for
30 min at ambient temperature. Subsequently, geranyl bromide
(0.95 mmol) was added dropwise under Ar atmosphere and under
exclusion of light and moisture. The reaction mixture was stirred
for 60 h at rt. Then, the salt was filtered off and washed with
CH2Cl2 (2”25 mL); the filtrate and washings were combined and
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partitioned between H2O (30 mL) and CH2Cl2 (40 mL) in a separation
funnel. The organic phase was pooled, and the aqueous phase was
washed with CH2Cl2 (2”25 mL). The combined organic layers were
dried (Na2SO4), filtered, and evaporated on a rotary evaporator. Re-
sidual DMF was removed by repeated co-evaporation with CH2Cl2
and subsequent drying in high vacuum. Column chromatography
(SiO2 60H, column: 5”6.5 cm, CH2Cl2/MeOH 95:5) gave the title
compound as a colorless solid (140 mg, 0.25 mmol, 40%).
ly, CH2Cl2/MeOH 9:1 or 95:5, respectively). Evaporation of the corre-
sponding main zone gave the N(1)-prenylated salts 9a-c.
6-Amino-9-[2-(2-ethoxycarbonyl-ethyl)-6-hydroxymethyl-2-
methyl-tetrahydro-furo[3,4-d][1,3]dioxol-4-yl]-1-(3-methyl-but-2-
enyl)-9H-purin-1-ium bromide (9a, NL_5.1.11.0,[23]): The product
was obtained as a white solid (0.44 g, 0.95 mmol, 63%).
6-Amino-1-(3,7-dimethyl-octa-2,6-dienyl)-9-[2-(2-ethoxycarbonyl-
ethyl)-6- hydroxymethyl-2-methyl-tetrahydro-furo[3,4-d][1,3]di-
oxol-4-yl]-9H-purin-1-ium bromide (9b, NL_5.1.12.0,[23]): The
Ethyl 3-{4-hydroxymethyl-2-methyl-6-[6-oxo-1-(3,7,11-trimethyl-
dodeca-2,6,10-trienyl)-1,6-dihydro-purin-9-yl]-tetrahydro-
furo[3,4-d][1,3]dioxol-2-yl}propionate (8c, NL_6.1.13.0,[23]): To a so-
lution of anhydrous, stereochemically pure compound (1R)-4
(0.2 g, 0.51 mmol) in dry DMF (3 mL), K2CO3 (178 mg, 1.32 mmol)
was added. The suspension was stirred at ambient temperature for
30 min. Then farnesyl bromide (0.16 mL, 0.56 mmol) was added
dropwise under Ar atmosphere. The reaction mixture was stirred
for 24 h at rt under exclusion of light and moisture. Thereupon,
the suspension was filtered, and the salt was washed repeatedly
with CH2Cl2 (2”25 mL). Filtrate and washings were evaporated on
a rotary evaporator, and the oily residue was dried in high vacuum.
The raw product was purified by column chromatography (SiO2
60H, column: 6.5”11 cm, CH2Cl2/MeOH 97:3). Evaporation of the
main zone gave the title compound as a colorless oil (175.1 mg,
0.29 mmol, 57%).
product was obtained as
1.48 mmol, 58%).
a slightly yellowish solid (0.78 g,
6-Amino-9-[2-(2-ethoxycarbonyl-ethyl)-6-hydroxymethyl-2-
methyl-tetrahydro-furo[3,4-d][1,3]dioxol-4-yl]-1-(3,7,11-trimeth-
yl-dodeca-2,6,10-trienyl)-9H-purin-1-ium bromide (9c, NL_
5.1.13.0,[23]): The product was obtained as a slightly orange oil
(0.98 g, 1.64 mmol, 67%).
General Procedure for the Dimroth rearrangements of com-
pounds 9a–c:[16,17] Compounds 9a–c (0.7 mmol, each) were sus-
pended in Me2NH–MeOH (1m, 4.5 mL) and stirred for 20 h at ambi-
ent temperature (TLC monitoring). Subsequently, the correspond-
ing reaction mixtures, containing compounds 10a, 10b, or 10c,
were concentrated on a rotary evaporator and then in high
vacuum. The residues were purified by column chromatography
(SiO2 60H, columns: 5”7 cm, 6.5”12.5 cm, and 6.5”10.5 cm; sol-
vent systems: CH2Cl2/MeOH, 98:2, CH2Cl2/MeOH, 95:5). The main
fractions were pooled and evaporated to give slightly colored oils
of 10a–c.
Repetition of the formerly described experiment at elevated
temperatures yielding 8c and 9-{2,3-O[(1R)-4-ethoxy-1-methyl-4-
oxobutylidene]-ß-d-ribofuranosyl}-6-{[(2E,6E,9E,13E,17E)-
3,7,11,11,14,18,22-heptamethyltricosa-2,6,9,13,17,21-hexaen-1-
yl]oxy}-9H-purine (12, via nonisolated 8d): To a solution of anhy-
drous, stereochemically pure compound (1R)-4 (0.15 g, 0.51 mmol)
in dry DMF (4.8 mL), K2CO3 (140 mg, 1.32 mmol) was added. The
suspension was heated to 508C for 30 min and then cooled to
408C. Then, farnesyl bromide (0.12 mL; 0.42 mmol) was added
dropwise stirred at ambient temperature for 30 min. Subsequently,
a second portion of farnesyl bromide (0.16 mL, 0.38 mmol) was
added dropwise under Ar atmosphere. The reaction mixture was
stirred for another 24 h at rt under exclusion of light and moisture.
Then, CH2Cl2 (6 mL) was added; the suspension was filtered, and
the salt was washed repeatedly with CH2Cl2 (2”25 mL). Filtrate and
washings were evaporated on a rotary evaporator, and the oily res-
idue was dried in high vacuum. The raw product was purified by
column chromatography (SiO2 60H, column: 6.5”10 cm; 1. main
zone, CH2Cl2/MeOH; 94:6; v/v; 2. main zone, MeOH, 0.3 bar). From
the faster-migrating main zone, compound 8c was isolated as a col-
orless oil (128 mg, 0.214 mmol, 56%). The compound was identical
with an authentic sample of compound 8c in all other respects
(1H NMR, 13C NMR, HR ESI MS). From the slower-migrating zone,
the title compound 12 was isolated as a colorless oil (115 mg,
0.142 mmol, 37%).
Ethyl 3-{4-hydroxymethyl-2-methyl-6-[6-(3-methyl-but-2-enyla-
mino)-purin-9-yl]-tetrahydro-furo[3,4-d][1,3]dioxol-2-yl}-propio-
nate (10a, NL_5.1.61.0): The product was obtained as a colorless
oil (0.21 g, 0.46 mmol, 62%).
3-{4-[6-(3,7-Dimethyl-octa-2,6-dienylamino)-purin-9-yl]-6-hydrox-
ymethyl-2-methyl-tetrahydro-furo[3,4-d][1,3]dioxol-2-yl}-pro-
pionsäureethylester (10b, NL_5.1.62.0,[23]): The product was ob-
tained as a colorless oil (0.18 g, 0.34 mmol, 23%).
Ethyl
3-{4-hydroxymethyl-2-methyl-6-[6-(3,7,11-trimethyl-
dodeca-2,6,10-trienylamino)-purin-9-yl]-tetrahydro-furo[3,4-d]
[1,3]dioxol-2-yl}-propionate (10c, NL_5.1.63.0,[23]): The product
was obtained as a colorless oil (0.47 g, 0.62 mmol, 38%).
Biological methods
Cell lines and culture conditions: In vitro experiments were per-
formed using the human astrocytoma/oligodendroglioma GOS-3
cells (DSMZ GmbH, Braunschweig, Germany), the rat malignant
neuro-ectodermal BT4Ca cells (a kind gift from Dr. Nadine John,
Hannover Medical School, Hannover, Germany), as well as the
human acute monocytic leukemia cell line THP-1 (DSMZ GmbH,
Braunschweig, Germany). The cells were cultured in 90% RPMI
1640 medium supplemented with 10% fetal bovine serum (FBS),
100 UmLÀ1 penicillin, and 0.1 mgmLÀ1 streptomycin, and were
maintained at 378C in a humidified atmosphere (5% CO2, 95% air)
as described earlier.[1,2]
General procedure for the prenylation of the adenosine-O-2’,3’-
ketal 3: Anhydrous compound 3 (0.59 g, 1.5 mmol) was dissolved
in anhydrous and amine-free DMF (4 mL). To the solution was
added BaCO3 (0.89 g, 4.5 mmol). Under Ar atmosphere, isopentenyl
bromide, geranyl bromide or farnesyl bromide were added drop-
wise (1.95 mmol each). The reaction mixtures were stirred o/n at rt
under exclusion of moisture and light. Subsequently, the salt was
removed by filtration through a Celite pad. The corresponding fil-
trates were evaporated on a rotary evaporator, and the residues
were then dried in high vacuum to give a slightly red foam. Tritura-
tion with CH2Cl2 (50 mL) afforded an off-white solid which was iso-
lated by filtration. Purification was performed by column chroma-
tography (SiO2 60H, column: 5”8.5 cm or 6.5”10.5 cm, respective-
Determination of viability/survival of 5-fluorouridine and deriva-
tives: 96-well plates (BD Falcon, Becton Dickinson GmbH, Heidel-
berg, Germany) were seeded with 1.5”104 GOS-3, 5”103 BT4Ca, or
3”104 THP-1 cells. After 24 h, the medium was changed and differ-
ent concentrations of 5-fluorouridine or its derivatives 5, 6, 8c,
were tested at concentrations of 1.56, 3.12, 6.25, 12.5, 25, or 50 mm.
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After incubation for 48 h, viability/cytotoxicity was measured using
PrestoBlue reagent (Invitrogen-Life Technologies GmbH, Darmstadt,
Germany) as described earlier.[1,2] PrestoBlue was added to the cells
into the culture medium and 30 min, 1 h, 2 h, or 3 h after addition
of PrestoBlueꢁ, the optical density (OD) was measured at 570 nm
and 600 nm (as reference) with a SUNRISE ELISA-reader (Tecan,
Salzburg, Austria). Results are expressed in %survival (OD 570/
600 nm of samples ” 100 / OD 570/600 nm of control without sub-
stances). As control (100% viability=0% cytotoxicity), cells were
cultured with medium alone. To evaluate the cytotoxic effect on
macrophages, this procedure was repeated with 5”103 per well of
phorbol-12-myristate-13-acetate (PMA; 100 ngmLÀ1)-differentiated
(48 h) human THP-1 macrophages, which were treated (48 h) with
different concentrations of the substances under test.
software (Scion ImageJ, National Institutes of Health, Bethesda,
USA).
Statistical analysis: Results are presented as meansÆstandard
error of the mean (SEM). Depending on the mode of distribution,
statistical procedures were performed by the Mann–Whitney U test
or by the unpaired Student’s t-test, using SigmaPlot software
(Systat Software, Inc, Chicago, USA); p<0.05 was chosen for statis-
tical significance.
Acknowledgements
The authors thank the following University of Osnabrück and
Marburg members: Marianne Gather Steckhan for the NMR
measurements, Dr. Stefan Walter for HR ESI MS, Anja Schuster for
the elemental analyses, Petra Bçsel for formulae drawings, Hen-
ning Eickmeier and Holger Heine for the preparation of Figure 3
(Univerity of Osnabrück); Andrea Cordes, Elke Vçlck–Badouin
(University of Marburg); Diana Yudin and Anna Diestel (University
of Osnabrück) for valuable technical assistance; as well as Ellen
Essen and Gabriella Stauch (University of Marburg) for prepara-
tion of the manuscript. The authors also gratefully acknowledge
financial support by the Bundesministerium für Wirtschaft, Ger-
many (FKZ: KF 2369401 SB9 and FKZ 2369501 SB9). Moreover,
the authors thank Prof. Dr. Uwe Beginn for excellent laboratory
facilities at the University of Osnabrück.
Apoptosis assay: Human GOS-3 cells or rat BT4Ca were seeded in
96-well plates at a density of 1.5”104 and 5”103 per well, respec-
tively. After 24 h, the medium was changed, and the substances
under test were added at various concentrations as indicated.
Apoptotic cells were identified by YO-PRO-1 (1 mm) in combination
with the Hoechst 33342 nuclear staining dye (5 mgmLÀ1; Mobitec
Company, Gçttingen, Germany). The total number of cells (Hoechst
33342+ nuclei) and apoptotic cells (YO-PRO-1+ nuclei) was counted
using an inverse fluorescence microscope Eclipse TS100 (Nikon
GmbH, Düsseldorf, Germany) equipped with a camera AxioCam
MRc (Carl Zeiss Microscopy GmbH, Gçttingen, Germany) and a com-
puter-assisted morphometry system AxioVision 4 (Carl Zeiss Micros-
copy GmbH), and the percentage of apoptotic cells to the total
cells was then calculated. Additionally the % of total cell count
after 48 h treatment was calculated using the total number of cells
containing Hoechst 33342+ nuclei, considering the negative con-
trol without treatment as 100%.[24]
Keywords: antitumor agents · cytotoxicity · glioblastomas ·
ketal lipophilicity · nucleolipids · prenylation
SDS-PAGE and western blotting: GOS-3 and BT4Ca cells were
scraped off in radioimmunoprecipitation assay (RIPA) buffer pH 7.5
(Cell Signaling Technology Europa, Leiden, The Netherlands). An ali-
quot was used for protein quantification using Pierceꢁ BCA Pro-
tein Assay Kit (Pierce Biotechnology, Rockford, USA), and after addi-
tion of sample buffer, pH 8.3, boiled at 958C (10 min). Samples
were stored at À208C. Afterwards, electrophoresis was done using
NuPAGE Novex 4–12% Bis-Tris precast polyacrylamide gels (Life
Technologies GmbH, Darmstadt, Germany) and a loading of 30 mg
total protein per lane. Additionally, prestained peqGOLD Protein-
Marker VI (PEQLAB Biotechnologie GmbH, Erlangen, Germany) was
used. Blotting was performed with wet/tank blotting systems (Bio-
Rad Laboratories GmbH, München, Germany) and nitrocellulose
Amersham Hybond-ECL membranes (GE Healthcare Europe GmbH,
Glattbrugg, Switzerland) for enhanced chemiluminescence (ECL).
Protein transfer was done at 0.6 mAcmÀ2 (o/n, rt) with transfer
buffer containing 10% MeOH. Nonspecific sites were blocked with
Tween 20-supplemented Tris-buffered saline (TTBS) and 5% milk
(150 mmNaCl, 10 mmTris/HCl, pH 7.6; Tween-20 0.1%; 5% low-fat
dried milk; 1 hour, rt). Rabbit polyclonal antibodies directed against
p62/SQSTM1 (P0068, 1:500, Sigma–Aldrich); rabbit polyclonal anti-
bodies against active caspase-3 (ab2302, 1:200, Abcam, Cambridge,
UK); BAX (Ab7977, 1:1000, Abcam); PCNA (13110, 1:1000, New Eng-
land Biolabs, UK); and a-tubulin (ab4074, 1:1000, Abcam) were
used diluted in blocking buffer, o/n, 48C. An anti-rabbit IgG perox-
idase conjugate (1:3000, GE Healthcare Europe GmbH, Germany) in
blocking buffer; 1 h, rt) and the chemiluminescence ECL detection
kit, AceGlow (PEQLAB Biotechnologie GmbH) were used for detec-
tion. A FUSION-FX7 system (PEQLAB Biotechnologie GmbH) served
for documentation. Washing steps after every incubation period
were performed using TTBS. Signals were quantified by computer
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