Synthesis and cytostatical evaluation of cytidine- and adenosine-5'-hexadecylphosphate and their phosphonate analogs

Article abstract of DOI:10.1016/S0009-3084(97)00074-1

Four phospholipid conjugates containing the non-cytotoxic nucleosides cytidine and adenosine were prepared by condensation reactions, and their cytotoxic activity was tested in vitro against the human immortalized mammary epithelial cell H184 A₁N₄, the human mammary tumor cells MaTu and MCF7 and the B lymphoblast cell line Daudi. The synthesized compounds showed considerable activity towards H184 A₁N₄, MaTu and Daudi cells, but they were not effective against MCF7 cells. The phosphorus moiety - either monophosphate or monophosphonate - does not influence the effectiveness of the phospholipid derivatives in the case of the solid tumor cell lines and H184 A₁N₄. The leukemic Daudi cell line is strongly sensitive towards the different types of ester as well as to the type of the nucleoside component. Adenosine-5'-hexadecylphosphate proved to be the most potent compound among the substances prepared (IC₅₀: 9.0 μmol).

Full text of DOI:10.1016/S0009-3084(97)00074-1

Chemistry and Physics of Lipids
90 (1997) 143–149
Short communication
Synthesis and cytostatical evaluation of cytidine- and
adenosine-5%-hexadecylphosphate and their phosphonate analogs
H. Brachwitz a,*, J. Bergmann a, Y. Thomas b, W.E. Berdel b, P. Langen a, T. Wollny b
a Max-Delbru¨ck-Centrum fu¨r Molekulare Medizin, R.-Ro¨ssle-Str. 10, 13125 Berlin-Buch, Germany
b Freie Uni6ersita¨t Berlin, Uni6ersita¨tsklinikum Benjamin Franklin, Abt. Ha¨matologie and Onkologie, Berlin, Germany
Received 9 May 1997; received in revised form 1 September 1997; accepted 2 September 1997
Abstract
Four phospholipid conjugates containing the non-cytotoxic nucleosides cytidine and adenosine were prepared by
condensation reactions, and their cytotoxic activity was tested in vitro against the human immortalized mammary
epithelial cell H184 A1N4, the human mammary tumor cells MaTu and MCF7 and the B lymphoblast cell line Daudi.
The synthesized compounds showed considerable activity towards H184 A1N4, MaTu and Daudi cells, but they were
not effective against MCF7 cells. The phosphorus moiety—either monophosphate or monophosphonate—does not
influence the effectiveness of the phospholipid derivatives in the case of the solid tumor cell lines and H184 A1N4. The
leukemic Daudi cell line is strongly sensitive towards the different types of ester as well as to the type of the nucleoside
component. Adenosine-5%-hexadecylphosphate proved to be the most potent compound among the substances
prepared (IC50: 9.0 vmol). © 1997 Elsevier Science Ireland Ltd.
Keywords: Phospholipids; Nucleoside-phospholipid conjugates; Antiproliferative activity
1. Introduction
as well as simple alkanols were found to be cyto-
statically active in vitro (Brachwitz et al., 1990a,
1996, 1997). In the latter case the activity strongly
depends on the length of the alkyl chain. The
C16-derivatives (cytidine-5%- and adenosine-5%-hex-
adecylphosphonophosphate 1d and 2d) were
found to be the most potent compounds. The
mechanism of their action is not clearly under-
stood but since they proved to be active substrates
Structurally modified analogs of the naturally
occurring cytidine- and adenosine-5%-diphosphate
diacylglycerol, in which the diacylglycerol (DAG)
component is replaced by modified alkylglycerols,
* Corresponding author. Tel./fax: +49 30 9406 3347;
e-mail: hbrachw@mdc-berlin.de
0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-3084(97)00074-1

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H. Brachwitz et al. / Chemistry and Physics of Lipids 90 (1997) 143–149
Fig. 1. Chemical structure of the newly synthesized nucleoside-5%-hexadecyl-monophosphates and -phosphonates (1 a,b; 2 a,b) and
the corresponding diphosphates and phosphonophosphates (1 c,d; 2 c,d).
of the phosphatidylinositol (PI) synthase (Brach-
witz et al., 1990b), the antiproliferative activity
might be due to the cellular formation of cyto-
toxic PI analogs according to the equation: nu-
phosphonophosphates, cannot be the cause of the
antiproliferative effects (Fig. 1).
cleoside
diphosphate-DAG-analog+inositol[
2. Experimental procedures
PI-analog+nucleoside monophosphate.
In this context it was of interest whether the
corresponding cytidine-5%- and adenosine-5%-decyl-
monophosphates (1a, 2a), which cannot be con-
verted into PI analogs according to the described
pathway will also exhibit any cytostatic activity.
In addition, the structurally related cytidine- and
2.1. Material and methods
Hexadecylphosphate was obtained by reaction
of hexadecyl alcohol and phosphorus oxychloride
in hexane in the presence of triethylamine accord-
ing to known procedures (Mangold and Paltauf,
1983). Hexadecylphosphonate was prepared by
the Michaelis–Arbuzov reaction of hexadecyl
bromide with triethyl phosphite (Lugovkin, 1957)
and conversion of the obtained diethyl ester into
the bis(trimethylsilyl) ester (Morita et al., 1978)
followed by hydrolysis of the latter. Pyridine was
distilled and stored over calcium hydride. Dry
ethanol-free chloroform was obtained by column
chromatography on Al2O3. All other solvents
were distilled prior to use. All reactions were
carried out at room temperature unless stated
otherwise. N4-benzoyl cytidine and N4-benzoyl-
2%,3%-O-isopropylidene cytidine were prepared ac-
adenosine-5%-hexadecylmonophosphonates
(1b,
2b) bearing a non-hydrolysable C–P bond resis-
tant to hydrolytic cleavage by phospholipase C
were synthesized. The present communication de-
scribes the preparation of these compounds and
their cytostatic activity in comparison with the
corresponding diphosphates and phosphonophos-
phates. It was found that both the monophos-
phates (1a, 2a) and the monophosphonates (1b,
2b) inhibit the cell growth of different types of cell
lines. These results indicate that in this case the
intracellular formation of cytotoxic PI analogs, as
assumed for corresponding diphosphates and

H. Brachwitz et al. / Chemistry and Physics of Lipids 90 (1997) 143–149
145
cording to known procedures. (Watanabe and
Fox, 1966; Holy and Pischel, 1974).
for 48 h. After incubation with the XTT labeling
mixture for 4 h at 37°C plates were measured in
an ELISA reader (SLT Labinstruments) with 450
nm and a reference wavelength of 620 nm.
The B lymphoblast cell line Daudi was grown
in RPMI medium containing 10% FCS. In this
case the cytostatic potency of the nucleoside-phos-
pholipid derivatives was determined with a cell
counter.
The concentration of the compounds resulting
in 50% inhibition of cell growth (IC50 value) was
estimated. The values are given as the mean +
SEM of three separate experiments.
The nucleoside-5%-hexadecylphosphates 1a and
2a gave clear solutions in water up to concentra-
tions of 10−4 mol/l. The nucleoside-5%-hexade-
cylphosphonates 1b and 2b are less soluble
showing slight turbidity at concentrations of 10−4
mol/l.
2.2. Analytical methods
Analytical thin-layer chromatography (TLC)
was carried out on Merck silica gel 60 F254 plates.
Phosphorus containing compounds were visual-
ized with Zinzade’s reagent modified according to
Beiss (Kates, 1972). Hexadecyl components were
detected by fluorescent spots (366 nm) on the
TLC-plate after spraying with 2,7-dichlorofluores-
cein in EtOH (0.2%). The nucleoside derivatives
could be visualized by ultraviolet absorption (254
nm). Column chromatography was performed
with Merck silica gel 60 (0.015–0.040 mm,
ASTM). The eluents are solvent a: CHCl3/MeOH/
NH4OH/H2O 70:30:4:4 (v/v/v/v) and solvent b:
CHCl3/MeOH/NH4OH/H2O 100:30:4:4 (v/v/v/v).
1H NMR spectra were recorded on a Varian
Gemini 300 spectrometer operating at 300 MHz,
31P NMR spectra on a UNITYplus-300 operating
at 122 MHz in DMSO-d6; chemical shifts (l) are
reported in ppm referenced to tetramethylsilane.
Electrospray ionization mass spectra (ESI MS)
were recorded in positive or negative ion mode on
a Varian MAT 95 mass spectrometer.
2.4. Syntheses
2.4.1. Cytidine-5%-hexadecylmonophosphate (1a)
Cytidine monophosphate (0.50 g, 1.55 mmol),
acetanhydride (20 ml) and pyridine (40 ml) were
stirred overnight. Water (25 ml) was added to the
ice-cold mixture and stirring was continued for
additional 2 h. After evaporation N4,2%,3%-tri-
2.3. In 6itro studies
acetyl-cytidinephosphate was obtained as
a
brownish oil (Rf: 0.85, solvent a), which was used
in the next stage without further purification. A
mixture of the peracetate and 2,4,6-triisophenyl-
sulfonyl chloride (1.86 g, 6.20 mmol) was dis-
solved in pyridine (30 ml). After complete
solution hexadecyl alcohol (750 mg, 3.1 mmol)
was added and the mixture was stirred for 16 h.
Water (2 ml) was added and the mixture was
stirred for additional 3 h. After evaporation of the
solvent, diethylether was given to the residue and
the suspension was filtrated. The filtrate was evap-
orated to dryness and the residue was chro-
matographed (solvent b) to give 357 mg (34%) of
N4,2%,3%-triacetyl-cytidine-5%-hexadecylphosphate
(Rf: 0.65, solvent a) which was stirred with 2 N
NH3-MeOH (20 ml) for 5 h to remove the acetyl
groups. After evaporation of the solvent the
residue was purified by column chromatography
(solvent a). After rechromatography with the
We used an immortalized (by benzpyrene), but
nontumorigenic human mamma epithelial cell line
(H184 A1N4) (obtained from Dr M. Stampfer,
Lawrence Berkeley Laboratory, University of
California, Berkeley, CA) and the human breast
cancer cell lines MaTu (Widmaier et al., 1974)
and MCF7.
The H184 A1N4 and the MaTu cell line were
grown in h-MEM/HAMS F12 (1:1, vol/vol)
medium as described in the literature (Bergmann
et al., 1994). MCF7 cells were cultured in RPMI
medium containing 10% FCS. The antiprolifera-
tive potency of the nucleoside derivatives was
determined using the XTT assay (Cell Prolifera-
tion Kit II, Boehringer, Mannheim). Cells were
seeded in microtiter plates (Costar) and incubated
for 48 h at 37°C and 5% CO2. The compounds
were added, and the culture was continued further

146
H. Brachwitz et al. / Chemistry and Physics of Lipids 90 (1997) 143–149
same eluent, 275 mg (95%) of the title product
(Rf: 0.45, solvent a) was obtained.
2.4.3. Adenosine-5%-hexadecylphosphate (2a)
Adenosine (535 mg, mmol), 2,2-
2
Mp: \230°C (decomp.).
dimethoxypropane (3 ml) and p-toluenesulfonic
acid (500 mg) were stirred in acetone (30 ml).
After 2 h an ice-cold 10% aqueous NaHCO3-solu-
tion was poured into the reaction mixture and
stirring was continued for 30 min. The mixture
was evaporated to dryness and the residue was
extracted with acetone in a Soxhlett apparatus.
The product was recrystallized from acetone to
give 405 mg (66%) of 2%,3%-isopropylidene
adenosine (Rf: 0.86, solvent a). Hexadecylphos-
phate (483 mg, 1.5 mmol) and 2,4,6-triisophenyl-
sulfonyl chloride (1.80 g, 6.0 mmol) were stirred
in pyridine (25 ml) for 30 min. To this solution
2%,3%-isopropylidene adenosine (305 mg, 1 mmol)
was added and stirring was continued for 16 h.
Then 10 ml of water was given to the reaction
mixture and after 1 h it was evaporated to dry-
ness. The remaining solid was extracted with di-
ethyl ether (3×20 ml) and the organic solution
obtained was evaporated. The residue was chro-
matographed (solvent a) and after rechromatogra-
ESI MS (negative ion mode): 546.3 [M−H]−.
1H NMR: l 0.85 (t, J=6.5 Hz, 3 H; Me-alkyl
chain); 1.00–1.30 (m, 26 H; alkyl chain); 1.40–
1.60 (m, 2 H; PO-CH2-CH2-alkyl chain); 3.66 (dt,
J=6.5 and 6.5 Hz, 2 H; PO-CH2-alkyl chain);
3.82 (mc, 1 H; 4%-CH); 3.85–4.00 (m, 4 H; 2%,3%-
CH, 5%-CH2); 5.76 (d, J=7.5 Hz, 1 H; 5-CH);
5.78 (d, J=2.5 Hz; 1%-CH); 7.86 (d, J=7.5 Hz;
6-CH).
31P NMR: l 0.694.
2.4.2. Cytidine-5%-hexadecylphosphonate (1b)
N4-benzoyl-2%,3%-O-isopropylidene
cytidine
(Holy and Pischel, 1974) (300 mg, 0.78 mmol) was
reacted with hexadecylphosphonate (309 mg, 1.01
mmol) in the presence of 2,4,6-triisopropylphenyl-
sulfonyl chloride (1.21 g, 4.0 mmol) in pyridine
(10 ml) according to the procedure described for
the synthesis of compound 2 to give 420 mg of a
solid which was purified by column chromatogra-
phy (solvent a). 173 mg (33%) of N4-benzoyl
2%,3%-O-isopropylidene cytidine-5%-hexadecylphos-
phonate (Rf: 0.87, solvent a) was isolated as white
solid which was stirred with 3 N NH3-MeOH for
5 h to remove the benzoyl group. After evapora-
tion and column chromatography (solvent a) and
rechromatography (solvent b) 131 mg (90%) of
2%,3%-O-isopropylidene cytidine-5%-hexadecylphos-
phonate (Rf: 0.74, solvent a) was obtained as a
white powder (mp: \230°C decomp.). Then this
compound (100 mg, 0.175 mmol) was stirred in
80% aqueous acetic acid at 70–80°C. After 2 h
the reaction mixture was evaporated and the
residue was chromatographed (solvent a) and
rechromatographed (solvent b) to give 60 mg
(56%) of the title product (Rf: 0.42, solvent a) as a
white powder.
phy
(solvent
b)
390
mg
(64%)
of
2%,3%-isopropylidene adenosine-5%-hexadecylphos-
phate (Rf: 0.80, solvent a) was obtained.
This compound (350 mg, 0.57 mmol) was
heated in 100 ml 80% aqueous acetic acid at
70–80°C for 2 h. After evaporation to dryness the
residue was chromatographed (solvent a) and
rechromatographed (solvent b) to give 167 mg
(51%) of the title product (Rf: 0.57, solvent a) as a
white powder (identical to the compound men-
tioned in the literature (Shiraishi et al., 1994;
Hynie and Smrt, 1978) which was prepared differ-
ently).
ESI MS (positive ion mode): 572.3 [M+H]+
1H NMR: l 0.85 (t, J=6.5 Hz; 3 H; Me-alkyl
chain); 1.10–1.16 (m; 26 H; alkyl chain); 1.43 (mc;
2 H; PO-CH2-CH2-alkyl chain); 3.59 (dt, J=6.5
and 6.5 Hz; 2 H; PO-CH2-alkyl chain); 3.86–3.90
(m; 2H; 5%-CH2); 4.01 (dt, J=3 and 3.5 Hz; 1 H;
4%-CH); 4.19 (dd, J=5 and 3 Hz; 1 H; 3%-CH),
4.56 (ddd, J=5.5 and 5 and 4 Hz; 1 H; 2%-CH),
5.38–5.70 (m; 2H; 2%- and 3%-OH); 5.91 (d, J=5.5
Hz; 1 H; 1%-CH); 8.13 (s; 1 H; 2-CH), 8.44 (s; 1H;
8-CH).
ESI MS (positive ion mode): 532.4 [M+H]+.
1H NMR: l 0.85 (t, J=6.5 Hz, 3 H; Me-alkyl
chain); 1.00–1.0 (m, 30 H; alkyl chain); 3.80–4.00
(m, 5 H; 2%,3%,4%-CH, 5%-CH2); 5.72 (d, J=7.5 Hz,
1 H; 5-CH); 5.78 (d, J=2.5 Hz; 1%-CH); 7.81 (d,
J=7.5 Hz; 6-CH).
31P NMR: l 24.32.
31P NMR: l 0.529.

H. Brachwitz et al. / Chemistry and Physics of Lipids 90 (1997) 143–149
147
2.4.4. Adenosine-5%-hexadecylphosphonate (2b)
According to the procedure described for the
synthesis of compound 6a hexadecylphosphonate
(460 mg, 1.5 mmol), 2,4,6-triisopropylphenylsul-
fonyl chloride (1.80 g, 6.0 mmol) and 2%,3%-iso-
propylidene adenosine (305 mg, 1.0 mmol) were
reacted to give 157 mg (28% from 2%,3%-isopropyli-
dene adenosine) of the title product (Rf: 0.51,
solvent a).
For the preparation of adenosine-5%-hexade-
cylphosphate 2a (Shiraishi et al., 1994; Hynie and
Smrt, 1978) and adenosine-5%-hexadecylphospho-
nate 2b the 2%,3%-hydroxy groups of adenosine 5
were protected by transformation into the corre-
sponding isopropylidene ketal. The amino group
was left unprotected since initial experiments re-
vealed that subsequent condensation with hexade-
cylphosphate or hexadecylphosphonate proceeds
exclusively at the 5%-hydroxy group to give 2%,3%-
isopropylidene adenosine-5%-hexadecylphosphate
and 2%,3%-isopropylidene adenosine-5%-hexade-
cylphosphonate, respectively. Deprotection of the
hydroxy groups with hot aqueous acetic acid
yielded the desired nucleoside adducts 2a, 2b in
about 20% overall yield.
ESI MS (negative ion mode): 554.2 [M−H]−.
1H NMR: l 0.85 (t, J=6.5 Hz, 3 H; Me-alkyl
chain); 1.00–1.50 (m, 30 H; alkyl chain); 3.70–
3.90 (m; 2H; 5%-CH2); 4.00 (dt, J=3 and 3.5 Hz;
1 H; 4%-CH); 4.28 (dd, J=5.5 and 3 Hz; 1 H;
3%-CH), 4.58 (ddbr, J=5.5 and 5.5; 1 H; 2%-CH),
5.88 (d, J=5.5 Hz; 1 H; 1%-CH); 8.13 (s; 1 H;
2-CH), 8.43 (s; 1H; 8-CH).
31P NMR: l 22.58.
3.2. Cytostatic acti6ity
The four compounds 1a, 1b, 2a and 2b were
tested for their cytostatic activity against four
types of cell lines, the human immortalized mam-
mary epithelial cell line H184 A1N4, the human
mammary tumor cell lines MaTu and MCF7 and
the B lymphoblast cell line Daudi. For compari-
son earlier results of the corresponding diphos-
phates 1c, 2c and phosphono-phosphates 1d, 2d
are included. In Table 1 the IC50 values (the
concentration of the compounds resulting in 50%
inhibition of cell growth) are shown.
The results obtained show that the monophos-
ph(on)ates 1a,b and 2a,b also exhibit antiprolifer-
ative potency towards the H184 A1N4 and MaTu
cells as already reported for the corresponding
diphosphates and phosphonophosphates 1c,d and
2c,d.
The H184 A1N4 and MaTu cells were sensitive
against all prepared monophosphate nucleoside
adducts 1a,b and 2a,b whereas the MCF7 cells
were not affected by any of these phospholipid
adducts in a concentration as high as 100 vM. In
the case of the H184 A1N4 cells all compounds
showed an inhibition level in the same range (ca.
40 vmol) independent of the nucleoside present in
the phospholipid derivatives and the phosphorus
moiety, monophosphate or monophosphonate.
However, the MaTu cell line was more sensitive
to the adenosine adducts (2a,b) in comparison
3. Results and discussion
3.1. Chemistry
Cytidine-5%-hexadecylphosphate 1a was pre-
pared by conversion of cytidinemonophosphate
into its triacetate and subsequent esterification
with hexadecyl alcohol in the presence of DCC or
TPS. After chromatographic purification the pro-
tective groups were removed with methanolic am-
monia to give the desired product with about 30%
overall yield.
Cytidine-5%-hexadecylphosphonate 1b was pre-
pared starting from cytidine. In a first step the
amino group was protected by benzoylation
(Watanabe and Fox, 1966) and the 2%,3%-hydroxy
groups were transformed into the corresponding
isopropylidene ketal to give N4-benzoyl-2%,3%-O-
isopropylidene cytidine. This intermediate was
condensed with hexadecylphosphonate similarly
to the procedure described for the synthesis of the
phosphate 1a yielding N4-benzoyl-2%,3%-O-iso-
propylidene
cytidine-5%-hexadecylphosphonate.
The desired phospholipid 1b was obtained after
removal of the protective groups, which was
achieved by subsequent reaction with methanolic
ammonia to deprotect the amino function and hot
aqueous acetic acid to cleave the isopropylidene
group.

148
H. Brachwitz et al. / Chemistry and Physics of Lipids 90 (1997) 143–149
Table 1
IC50 values (concentration of the compounds resulting in 50% inhibition of cell growth) of the phospholipid derivatives 1a–d and
2a–d against different cell lines
1a
1b
1c
1d
2a
2b
2c
2d
H184
36.293.1
42.297.5
\100
43.794.0
42.791.4
\100
79.598.2
97.394.8
\100
16.290.7
14.891.6
\100
39.792.9
27.791.3
\100
37.893.1
29.093.3
96.094.0
57.392.2
25.093.0
36.092.0
\100
10.591.4
7.090.7
\100
MaTu
MCF7
Daudi
a
a
a
a
51.393.8
\100
—
—
9.090.5
—
—
a Not determined.
with the cytidine derivatives (1a,b), but the activ-
ity was also not influenced by the type of the
phosphorus-containing ester group. The behavior
of the compounds towards the two cell lines
MaTu and H184 A1N4 does not correlate with
results of earlier studies by our group in which the
phosphonophosphates of both nucleosides (1d,
2d) were found to be more effective than the
corresponding diphosphates (1c, 2c) (Brachwitz et
al., 1996, 1995, 1990b). As already mentioned it
was postulated that the effectiveness of the
diphosphates and phosphonophosphates is at-
tributable to the formation of cytotoxic PI-
analogs inside the cell according to the cellular
PI-synthesis (CDP-DAG+inositol“PI+CMP
catalyzed by PI-synthase). Since the correspond-
ing monophosph(on)ates cannot be substrates of
the PI-synthase, a different mechanism of action
must be responsible for the effectiveness of these
products. Furthermore, it is also questionable
whether the formation of PI-analogs is the deci-
sive factor for the antiproliferative activity of the
diphosphates 1c, 2c and the phosphonophos-
phates 1d, 2d. It was also presumed that the high
effectiveness of the phosphonophosphates as com-
pared to the diphosphates is the result of the
increased biostability of the P–C bond against
cleavage by phospholipase C. Apparently, this
stability of the P-C bond does not play an essen-
tial role for the antiproliferative activity of the
compounds 1a,b and 2a,b, which exhibit similar
activities. Thus, for the clarification of the real
mechanism of action further studies are necessary.
Studies with the Daudi cell line corroborate the
results found with the MaTu cell line that the
adenosine derivatives are more effective than the
corresponding adducts with cytidine as nucleoside
moiety. The compounds 2a and 2b exhibit a sig-
nificantly higher potency compared to 1a and 1b.
The question whether the high effectiveness is
linked to special physiological effects of adenosine
itself or its monophosphate is still unclear but in
preliminary studies the latter compounds were
found to be ineffective using a comparable con-
centration range. Furthermore, the phosphates of
both types of nucleosides 1a and 2a have a
markedly higher effectiveness than the corre-
sponding phosphonates 1b and 2b (see Table 1). It
should be mentioned that the adenosine-5%-decyl-
monophosphate 2a is the most potent compound
(IC50: 9 vmol) among the substances prepared.
Acknowledgements
This work was in part supported by the BMBF
through the grant 01GB9402. The biological mea-
surements of Ms I. Thun are gratefully acknowl-
edged.
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